text:
"Glacier National Park" Glacier National Park by Redlats
Glacier National Park Travel Guide: 10 reviews and 21 photos
Glacier National Park has numerous short hikes. We walked most of them. See the Must See Tips for brief explanations of the hikes.
There is a rest stop at Rogers pass that has a plaque honouring all the provinces. There is a nice view of the mountains at this high mountain pass. The road in front is the Trans-Canada Highway.
I took a picture of the sign at the rest stop so I could tell the mountains apart from each other (then I left my camera in my knapsack there, and drove away - lucky it was still there when we discovered it missing and drove back)
Glacier National Park is best known for Rogers Mountain pass. This pass was used by the CPR (Canadian Pacific Railway) to finish their railroad across Canada. However it had difficulty during winters (5 meters of snow in a year is not unusual). Now the railroad travels under the pass in a tunnel, and the Trans-Canada highway uses Rogers Pass
- Pros:A quiet National Park
- Cons:Too much snow in winter
- In a nutshell:Hikes and Railway history
Reviews (4)
The Rock Garden trail is a new trail that wanders through a 1000's years old rock avalanche. You need good footwear for... more travel advice
The Hemlock Grove trail is an interpretive boardwalk through an old growth forest featuring cedars and hemlocks. 0.4km... more travel advice
The abandoned Rails trail is a short trail that leaves from the Rogers Pass Centre. It follows the 100 year old... more travel advice
Loop Brook trail is a one mile (1.6km) loop around the Illecillewaet campground. This is a signed trail on a National... more travel advice
Comments (5)
Great photos and information on these interesting-looking trails in the Park! I am always fascinated by old railways that have tried to tame the mountains!
What a beautiful, peaceful place! Would love to go there one day. Good tips and great photos.
Great pics of a wonderful place.
Enjoyed the quiet and the nature of this park a few years ago
Way too few photos and tips! Way too few!
Redlats' Related Pages
Glacier National Park Travel Guide
Member Travel Pages
- "Glacier National Park, British Columbia"
- "Glacier National Park"
- "Finding glaciers"
- "Glacier National Park"
- "Roger's Pass"
- See All...
- Things to Do in Glacier National Park
- Warnings and Dangers in Glacier National Park
- See All...
"Live for today"
- Member Rank:
- 0 0 4 6 0
- Forum Rank:
- 0 0 6 0 3
- 541 Reviews
- 920 Photos
- Add Friend
- Follow
- Send Message
Badges & Stats in Glacier National Park
- 4 Reviews
- 7 Photos
- 0 Forum posts
- 5 Comments
- 1,428PageViews
- See All Stats
- See All Badges (53)
Have you been to Glacier National Park?Share Your Travels
Latest Activity in Glacier National Park
- Posted in Miscellaneous Forum "Re: More Rental Getaways? "
- updated a Glacier National Park Travel Page "Glacier National Park"
- Wrote a Review Rock Garden trail in Glacier National Park Things to Do
- Uploaded a Photo to "Rock Garden trail"
Photos in Glacier National ParkSee All Photos (7)
Top 10 Pages
- Top 5 Page for this destination Winnipeg Intro, 34 reviews, 59 photos, 2 travelogues
- Top 5 Page for this destination Mérida Intro, 29 reviews, 55 photos, 1 travelogue
- Top 5 Page for this destination Campeche Intro, 22 reviews, 57 photos
- Top 5 Page for this destination London Intro, 15 reviews, 49 photos
- Top 5 Page for this destination Progreso Intro, 14 reviews, 34 photos
- Top 5 Page for this destination Ruinas Uxmal Intro, 15 reviews, 27 photos, 1 travelogue
- Tucson Intro, 12 reviews, 22 photos, 1 travelogue
- Banff National Park Intro, 14 reviews, 20 photos, 1 travelogue
- Honolulu Intro, 17 reviews, 16 photos
- Yellowstone National Park Intro, 16 reviews, 17 photos


text:
'Afton' Waterproof Boot
Product not available
Have questions?
Chat with us or call 1.888.282.6060.
HideDetails & Care
Subtly slouched leather updates a waterproof, knee-high boot boosted with a Mush® Infused Insole for signature comfort and Spider365™ sole for incredibly versatile traction.
- Back zip closure.
- Approx. boot shaft height: 15 3/4"; 14" calf circumference.
- Waterproof leather and suede upper/synthetic lining/rubber sole.
- By Teva; imported.
- BP. Shoes.
'Afton' Waterproof Boot
Write a Review0 Reviews
Enter a valid first name.
Enter a valid last name.
Enter a valid email address in the format: firstname.lastname@example.com.
Close modal

subset: commoncrawl


text:
Skip to main content
Rosemount Town Pages
B² Design Build feels right at home
Rosemount police activity for April 18 - 24
County case gets its day in the Supreme Court
Photogenic Moose
Who wants to be a firefighter?
More Topics
crime and courts
Baseball: Irish falter in Farmington
Boys lacrosse: Focused on improvement
Boys track: Irish show progress at Wayzata
Girls lacrosse: Improved defense helps Rosemount to 3-1 start
Boys tennis: Irish get in the win column
More Topics
high school
Your weekend: Earth and Arbor Day is Saturday
RHS grad lives her literary dream
Running with purpose
Good deeds are in sight
Purple Rain will screen in Rosemount
More Topics
arts and entertainment
Wells Fargo Bank, vs Poppler
Wells Fargo Bank vs Galvez
ASSUME NAME Cuddle Mutt
Gayle Draus, 68
Rev. Arthur W. Holmes D.D, 90
Jay A.
Sylvia C. Tousignant Perron, 92
Carol J. Zeroth, 76
Letter: New ice rink would be a waste of money
Chuck Brooks' column: Like a visitor in his own home
Abby Linnett's column: Cord cut
Nathan Hansen's column: Sticky floors for everybody
Cleaning up UMore is an important step
More Topics
Magazine Rack
Weather Forecast
March 26, 2016
Nathan Hansen's column: The future is literally virtual
Back when I was a substantially younger person, the technology world was in a big rush to convince everyone virtual reality was our path toward some bright science-fiction future. We were all led to believe we were just months away from strapping on a pair of goggles and slipping...
opinion / columns
March 26, 2016 - 6:00am
Abby Linnett's column: Horse sense
A while back, I wrote a column raising the question of whether my horse is smarter than I am. I am going to take liberty here and say he's not. I am definitely smarter than my horse. I don't freak out when a chicken runs out in front of me. Sure, my horse has a much more highly...
opinion / columns
March 26, 2016 - 6:01am
Chuck Brooks' column: Ready for the operating table
If you're reading this before Good Friday, the wait continues. If you're reading this after Good Friday, it's over. If you're reading this on Good Friday, then it could be happening RIGHT NOW! I have mentioned my issue with my knee in the past. I'm not sure I've brought...
opinion / columns
March 26, 2016 - 5:59am
March 20, 2016
Nathan Hansen's column: Just wait until next year
The NCAA men's basketball tournament begins its annual takeover of our television screens and quasi-legal office wagering pools this week. Across the country, die-hard fans are painting their faces and chests and other areas I'd just as soon they kept to themselves and preparing...
opinion / columns
March 20, 2016 - 6:01am
Chuck Brooks' column: This game is something to shout about
"N 33! O 68! B 2! "BINGO!" When I was in my early teens, my church would regularly sponsor Bingo nights in our school gym. Normally, the stakes would be food like hams and turkeys. There were monetary awards, but food was the primary prize. Mom and Dad would take me, and I'd...
opinion / columns
March 20, 2016 - 6:00am
March 12, 2016
Nathan Hansen's column: Sorry about this, plant
I bought a plant last weekend, and if I'm honest I couldn't give you a good reason why. I mean, I have a reason. There was a spot in my living room where there had formerly been a plant, and there was no longer a plant there. I didn't have anything else to put there. And so,...
opinion / columns
March 12, 2016 - 6:01am
Abby Linnett's column: Watch your stuff
Last weekend, two Rosemount High School jazz bands went to the University of Minnesota for a jazz festival. I am in one of the bands and was looking forward to a fun day full of music and great experiences with great friends. Little did I know, the day would be more interesting than...
opinion / columns
March 12, 2016 - 5:59am
Chuck Brooks' column: The loved ones we've lost live on within us
Sitting down to write this week's column, I can't quite shake the news that has occurred in the last 48 hours. A friend of mine suddenly lost his brother at the age of 51, and in the last 24 hours, a well-known educator in our district for many years who had retired some years...
opinion / columns
March 12, 2016 - 6:00am
March 5, 2016
Chuck Brooks' column: Recently retired and already buying a cane
March has arrived! And the weather report for the next several months shows favorable signs for those ready for spring. After that last near 60-degree day last weekend, I'd think most are more than ready to open windows, walk outside and simply watch the world turn green again....
opinion / columns
March 5, 2016 - 6:01am
Nathan Hansen's column: Lucky it's almost bike season
Before this week I had used any number of excuses to skip going to the gym. I didn't go because I was tired, or because it had been a long day. Because I had a school board meeting to go to, or a deadline to meet. Sometimes I didn't go because it had been too long since I'd...
opinion / columns
March 5, 2016 - 6:01am
February 28, 2016
Abby Linnett's column: On skiing — and not skiing — in Colorado
This past week I have been on vacation in Colorado, missing school to ski in Breckenridge, a ski resort my family has been coming to for 11 years. This is a bit of a challenge for a high school senior, as I missed three days of school. When you're in elementary school, you can...
opinion / columns
February 28, 2016 - 6:01am
Chuck Brooks' column: Living life at retirement pace
Can you believe we're already in the final days of February? Where did this month go? It's been six-plus months since the school year began and eight months since I was in a classroom as a teacher. A year ago, the emotions were beginning to stir. How could I make the break after...
opinion / columns
February 28, 2016 - 6:00am
February 27, 2016
Share your thoughts on comprehensive plan
As Mayor of Rosemount, I consider one of my most important duties to be preserving and improving the quality of life in our community. Those are the qualities that people mention to me when they explain why they've moved here and why they intend to stay. In city government, we're...
opinion / columns
February 27, 2016 - 6:01am
February 20, 2016
Nathan Hansen's column: Put it all on the walls
There is a fine line, it seems, between being a collector, being an accumulator and being that guy you read about on the news when the police find him buried under piles of old newspaper and cats. Buy a bunch of stuff and display it around your house, and you're an aficionado....
opinion / columns
February 20, 2016 - 6:01am
Chuck Brooks' column: The man with the microphone
I am sure you've heard the phrase, he "was born with a silver spoon in his mouth." That definitely does not describe my entrance into this world. However, I sometimes think I was born with a microphone in my hand. If you know me, you know what I mean. If you only know me through...
opinion / columns
February 20, 2016 - 6:00am
« Newer
Page 2
Older »


text:
Elva E. Johnson, age 100, of Albert City passed away Friday, September 12, 2003, at the Pleasant View Home in Albert City. A funeral service will be held on Tuesday, September 16, at 2 p.m. at Our Savior's Lutheran Church in Albert City with Reverend David Bernhardt officiating. Burial will be in the Fairfield Township Cemetery. The visitation will be at the church starting at 12:30 p.m. until the time of the service on Tuesday. The Sliefert ~ Asmus Funeral Home of Albert City is in charge of the arrangements.
Elva Elizabeth Johnson, one of twelve children of John P. and Louise (Johnson) Moline, was born on April 29, 1903, in Fairfield Township, Buena Vista County. She walked two miles each day to country school in rural Albert City and completed the eighth grade. At the age of 17, she worked as a clerk at the Scandia Trading Company in Albert City and remained there until she got married two years later.
On April 21,1923, Elva was united in marriage to Arthur T. Johnson and this union was blessed with two daughters, Dorothy and Norma. She raised her family on a farm in rural Albert City, and for 30 years, helped her husband with all the tasks of farming. Later in life, she was a cook at the Pleasant View Home in Albert City. Elva enjoyed many hobbies including quilting and crocheting. But, what she enjoyed most of all, was raising her family and spending time with her grandchildren, great-grandchildren and great great-grandchildren.
Elva's strong Christian faith was important to her. She served the Lord with a willing heart, helping the church in anyway she could. She was a member of Our Savior's Lutheran Church in Albert City. She also belonged to the Mayview Club and the Community Club.
Preceding Elva in death were her parents; her husband, Arthur in 1984, and her eleven brothers and sisters.
Survivors including two daughters: Dorothy Nordlund and her husband, Roland of Albert City and Norma Smith and her husband, Paul of Storm Lake; twelve grandchildren; 23 great-grandchildren and eight great great-grandchildren.
Funeral Services for Anna Helen Roling, formerly of Early, will be held Saturday, September 13, at Sacred Heart Catholic Church at 10:30 a.m. with Father Patrick O'Kane and Deacon Dennis Luft officiating. Burial will be in the Sacred Heart Catholic Cemetery in Early. Farber and Otteman Funeral Home, Sac City, is in charge of arrangements.
Anna Helen Roling, age 91, of Early, passed away on Wednesday, September 10, 2003, at Loring Hospital, Sac City.
Anna was born in Dunlap, on May 5, 1912, the daughter of James and Jesse (Jamesiah Kennedy) Nelis. She grew up in Vail, where she was baptized and confirmed at the Catholic Church and attended St. Anne's Catholic School.
On July 30, 1935, Anna was united in marriage to Leo J. Roling. They were the parents of five children: Joan, Bill, Jim, Mary Kay, and John. They lived in Vail until moving to Early in the early 1940's. She was a member of the Sacred Heart Catholic Church in Early.
Anna was loving wife, mother, and grandmother. She enjoyed spending time with her family and enjoyed growing indoor plants.
She is survived by her daughter Joan Nath of Marion; two sons: Jim Roling and his wife Sheryl of Spencer, and John Roling of Madison, WI; son-in-law Jerry Mueller of Phoeniz; daughter-in-law Marlene Roling of Minneapolis, MN; 19 grandchildren; 31 great-grandchildren; one great-great grandson; three sisters-in-law: Alice Roling and Irene Roling Bathurst, both of Early and Cecilia Nelis of Oklahoma City, OK; and extended family.
Anna was preceded in death by her parents; husband Leo on March 4, 1996; son William; daughter, Mary Kay Mueller; a brother Jack Nelis; and sister Margarite Luft.


text:
Reviews - Westpoint Hotel2
102 Conway Road, Colwyn Bay, LL29 7LE
Price From £45View Rooms
Customer Reviews
Our reviews are from genuine LateRooms.com customers. We verify that the customer has stayed before leaving a review.
Very Good
86% would recommend it to a friend
Customer service
Food quality
Room quality
West point one night stay
Nice stay.
The owners were extremely friendly and very helpful. Shower and food was OK and fit for purpose
Read More Less
Westpoint Hotel
Very clean and comfortable room.
a good family run hotel
although advertised as licensed, there was no bar or residents lounge.
handy for all public transport, beaches and town shops.
Read More Less
Nice place
Nice place, very friendly hosts.
Quiet street, rooms very well ventilated. Great full English breakfast.
Stayed in 3 different places (Coventry, Colwyn Bay, Bristol) in our four day trip and this was the cheapest (for 4 adults) and the best place of those.
Read More Less
Great weekend stay!!!
Had a short weekend stay with my 4yr old daughter and mom and dad in separate rooms.
Couldn't have asked for better service, very welcoming and really helpful with local points of interest. The rooms where really clean, had decent views, great facilities and breakfast was nice. Even with my 4 yr old running round being playful the hotel staff couldn't do enough for us. Would definitely recommend a stay!!!.
Read More Less
Westpoint Hotel
Only stayed one night with friends room was clean and comfortable, nice breakfast and cheap price.
Extremely friendly
The welcome we received when we got to this hotel was wonderful.
The proprietors Jan and Mick are lovely people. Jan went out of her way to help us when we needed an item of uniform laundering returning the item clean and ironed whilst we were out.
The breakfast was beautiful, well cooked and plentiful. Nothing seemed to be too much trouble. We hope to stay again next year. Thank you
Read More Less
Good value
Clean room, warm and cosy and had a good nights sleep Hotel itself needs a bit of renovation and the garden view would have been nice if the garden was actually nice to look at.
But overall for what I paid I was happy and I would still stay again
Read More Less
very good customer service. Value for money
We were 5 people.
Good service and morning breakfast was good.
Read More Less
New Years Break
A bit pricey for what you get.
Drafty windows in the room we stayed in, door lock could be opened with any key and you could not lock it from the inside. Smell of damp throughout the hotel. Although the breakfast was included we had it only once during the 3 night stay - sad there was no option without it as the food wasn't great. Decent car park and wi-fi - that's about it.
Read More Less
- < previous
- 1
- 2
- 3
- page 1 of 3
- > next


text:
No Lockouts Here: Celebrating Amateur Hockey
Commentator Mark Phelps plays in a hockey league without a union or owners. It's called men's league hockey, and in the case of his team, it's a bunch of middle-aged guys playing for the love of the game.
You must be signed in to leave a comment. Sign In / Register
Please keep your community civil. All comments must follow the NPR.org Community rules and terms of use, and will be moderated prior to posting. NPR reserves the right to use the comments we receive, in whole or in part, and to use the commenter's name and location, in any medium. See also the Terms of Use, Privacy Policy and Community FAQ.


text:
- New Releases
- Top Sellers
- Coming soon
Early Onset Alzheimer's
by Megan Carnarius
Early-onset Alzheimer's occurs as a diagnosis when someone is under the age of 65. This is still considered rare and occurs in about five percent of all Alzheimer's cases. It is difficult to get a proper diagnosis at times because clinicians don't typically look for this disease when trying to figure out what is causing problems for this younger individual. Sometimes psychiatric ...Read More
Transformation of Consciousness
by David Bennett
When we transform our consciousness, we participate in the transformation of the world we know. Let us look through the fog to see what a transformation in consciousness is and promote its development, also let's examine catalysts and doorways we know of that produce a life long lasting transformation.
Because you have a hammer in your hand, doesn't mean you know how to build a hous...Read More
Stewart Pearce brings you an Oracle Reading for the month of May
Chris Alexandria helps you to create positive powerful Askfirmations.


text:
Vermont Supreme Court rules in dog custody caseThe Associated Press | April 25,2014The Vermont Supreme Court has ruled in a custody case involving an 11-year-old dog.
The court on Friday agreed with a lower court assigning ownership of the German wirehaired pointer to the husband.
The court said the only issue in the contested divorce was which spouse should receive Belle.
The husband is a veterinarian who takes the dog to work while the wife spends time walking the dog in the woods and caring for the animal.
The wife appealed to the Supreme Court saying the court erred by not considering giving the dog to both spouses in a joint arrangement.MORE IN This Just In
BARRE - A Vermont state representative has admitted to one count of negligent driving... Full StoryMONTPELIER The Vermont Fish & Wildlife Department is urging residents to avoid attracting... Full Story
MONKTON — A woman who occupied a tree to block pipeline construction... Full Story
- Most Popular
- Most Emailed


text:
Handmade Craft Ideas from 2010: A Year of Free Crafts
Bonus: Get our newsletter & special offers for free. We will not share or sell your email address. View our Privacy Policy
How to Make a Tea Invitation Card
We are adding the craft to your Craft Projects.
The project was added to your Craft Projects.
Click here to view your Craft Projects.
Change the way you look at Thanksgiving! This year, in addition to the usual turkey and stuffing feast, have friends and family over for a celebratory tea party. Learn how to make a tea invitation card from Candace Jedrowicz and get inspired before the festivities.
This paper craft is so easy to create because the inside and outside are printables! Use your imagination to add fun, personal touches to this invitation. Turn this tea party into a yearly tradition and give thanks for the love in your life. These easy invitations can be completed in an afternoon.
For a step-by-step photo tutorial of this project, check out Thanksgiving Tea Time Invitation.
Tags / Related Topics
Your Recently Viewed Projects
Images from other crafters
Love these projects? Get MORE delivered to your Inbox – FREE Click Here!
Report Inappropriate Comment
Are you sure you would like to report this comment? It will be flagged for our moderators to take action.
Thank you for taking the time to improve the content on our site.
What's Hot
- How to Make a Soda Can Sunflower
- Doily Angel
- How to Create a Magazine Purse
- How to Tie a Fabric Bow on Mason Jar
- How to Make Look of Forged Metal with Aluminum Foil Video
- Rolled Fabric Roses
- How to Make a Soda Bottle Birdhouse
- Coffee Filter Flowers Video
- How to Make Iron On Glitter Lettering
- How to Make Paper Plate Flowers
Something worth saving?
- SAVE all your favorite crafts
- ADD personal notes
- QUICKLY reference your crafts
Free eBooks
- 13 Spring Crafts: Easter Craft Ideas, Spring DIY Home Dcor, and More
- 17 Flower Craft Ideas: How To Make Paper Flowers, Easy Fabric Flowers and More
- 18 Projects Every Crafter Wants
- 23 Fabulous Picnic Craft Ideas + Restaurant Copycat Recipes
- 25 Cool Things to Do with Wine Bottles
- 26 Spring Crafts for Kids
- Easter Craft eBook: Blogger Edition
- Garden Crafts
- Hop into Spring: Easter Crafting and Recipe
- Trash to Treasure: 28 Recycled Crafts
Our Newest Projects & Articles
- It's A Boy Light-Up Card
- Monogrammed China Plate
- DIY Felt Flowers
- Baby Octopus Crochet Applique
- Happy Thoughts Printable Coloring Page
- Gathered Waist Summer Dress Pattern
- Glass Pedestal Flower Holders
- DIY Pebble Coasters
- Back-to-School Crayon Art
- Flower Swirl Doodle Pages


text:
Bessie Jane King Brooks was born Aug. 31, 1944, and departed this world to be with her Lord on Monday, March 23, 2009, in McMinnville, Ore. Bessie was raised and attended school in Kennett. She was married and moved away to Oregon.
She is survived by her children, Jessie Carter, Janet Horton and Joella Brooks, also of McMinnville, and her sisters, Jerri Long, of Oakland, Calif., and Georgia Kraft, of West Plains, Mo.
She was preceded in death by her parents Bessie King, of Mt. View, Mo., and George King, Jr., of Bunevista, Colo.; grandparent, Ethel and Tom Pierce and many more relatives of Kennett.
Memorial services to celebrate her life will be held in McMinnville.
In lieu of flowers donations are being accepted at the funeral home of Macy and Sons at 135 NE Evans St., McMinnville, Oregon 97128.
Bessie will be greatly missed by family and friends.


text:
Support England's 2018 World Cup bid REGISTER NOW!
July 13th, 2009
One of these men may feature at the 2018 World Cup Even though 2018 seems an awful long way off, imagine how amazing it would be to have a World Cup in England… For many of us, it's something we've never had the chance to experience, and more than 50 years after 1966, Pies thinks […]
Flashback: Southampton score one of the greatest team goals in football history
July 13th, 2009
Just a goal of absolute beauty and as fine an entry into Pies' canon of forgotten wonder goals as you could ever hope to see; King Keggy's involvement in it is key. And what a lovely Southampton kit that was. [Spotted @ Guardian]
A random football list: Top 40 magnificent moustaches
July 13th, 2009
Where did all the moustaches go? These days it's all bare upper lips and waxed chests. Shame. Get a load of this… Caesar Jenkyns Hail Caesar. The rugged Welshman was one of the hardest defenders of his era -Â he played for Woolwich Arsenal from 1895-96, and became the Gooners' first 'international' captain. Bob Holmes Elementary. […]
Sh*t Lookalikes: Landon Donovan and Vegeta from Dragon Ball Z
July 13th, 2009
OK, this is shit, but shit meaning good -Â Landycakes needs bigger hair and a meaner glare, but he's not far off. And any time I can squeeze a pixelated game character into Shit Lookalikes, I leap at the chance. If you've never played Dragon Ball Z, there's more on Vegata here. Got any more videogame […]
Video: Cristiano Ronaldo and Karim Benzema take part in first Real Madrid training session
July 13th, 2009
La Liga
How do Galacticos break the ice? By comparing pay packets? If so, C-Ron wins. And do you think Raul is jealous of the new young crop of superstars at his club? Would love to be a fly on the wall at one Madrid's pre-season training sessions.
Gold Cup highlights: Stuart Holden spares USA blushes with wonder goal against Haiti
July 12th, 2009
International football
The USA has never lost a group match in the history of the CONCACAF Gold Cup. That record was severely tested by Haiti this weekend, when only a brilliant Stuart Holden strike in injury time saved the pre-tournament favourites from an embarrassing defeat. Highlights of the 2-2 draw below: Despite the draw, the Haitians still […]
David Bentley hit in the face. Much sympathy out there?
July 12th, 2009
'I've got a bad feeling about this…' 'Just smile Daveed, everything will be fine…' So David Bentley was thumped in the face as he sat in a restaurant. Not nice for the player and especially nasty for his heavily preggo wife, who witnessed the attack. Even if there's a very small part of you that […]
MLS star calls referee a 'cheat' on Twitter
July 12th, 2009
Funny that Brian Ching, the first footballer we featured in our new series, Footballers Who Tweet, is now in a spot of trouble thanks to the social networking site. Ching Tweeted the following message yesterday, immediately after Houston Dynamo lost 2-1 to Seattle Sounders: Why was Ching so incensed? The ref had allowed a goal […]
Video FAIL: Sky Sports presenter says the C word
July 12th, 2009
Stockport c**t. I laughed. [Thanks to the Guardian's Sean Ingle for the spot, via his Twitter feed]
Who the hell does John Terry think he is?
July 12th, 2009
This from The Times: 'Though [John] Terry has had several injuries in recent seasons, Chelsea continue to insist he is "not for sale at any price". The club are, however, mindful of dealings with Terry over his last contract talks in 2007, when he signed a five-year deal. 'During those negotiations, Terry asked for a […]
Photo Gallery: Fulham beat Melbourne Victory 3-0 on pre-season tour
July 12th, 2009
After a shock midweek defeat to Gold Coast United, Fulham won the second game of their pre-season tour of Australia. Roy Hodgson's team, led by skipper Danny Murphy, strolled to a 3-0 win against Melbourne Victory (the A-League champions). Their goals were scored by Andy Johnson, from the penalty spot, Eddie Johnson and Erik Nevland. […]
Snapshot Classic: Trevor Brooking starts career at West Ham United
July 11th, 2009
An extremely fresh-faced Trevor Brooking (aged 17) poses in a West Ham shirt prior to the start of the 1965/66 season. [Photo: PA/Barratts/S&G and Barratts/EMPICS Sport] Brooking signed for the Hammers as a teenager after leaving Ilford County High School. He went on to make 635 first-team appearances over the course of 19 years. This […]
Video: Real Madrid unveil Karim Benzema
July 10th, 2009
Real Madrid
Another day, another expensive galactico paraded in Madrid… Karim Benezma didn't quite match the 80,000 screaming fans who came to see C-Ron, but he still drew almost 20,000 to the Bernabeu, which isn't bad for a player who is not an A-List baller – not yet anyway. No doubt Madrid fans love him already for […]
Steven Gerrard makes theatre debut
July 10th, 2009
[Photo: PA] Steven Gerrard trod the boards – for the first time, as far as I'm aware – at Liverpool's Empire Theatre last night. Liverpool's captain was in the audience to watch the opening night of 'One Night In Istanbul', a play about four Liverpool supporters who journeyed to Istanbul to watch the 2005 Champions […]
Spot the Baller: 10 July, 2009
July 10th, 2009
I think you know what to do by now. Name the ball-headed baller… [Click the image to enlarge. Photo: PA] Think you know who's celebrating with Becks? Leave a comment with your answer. Yesterday's answer: Darren Anderton Most of you recognised the fragile stick legs of Sicknote. But top of the class is Liam FP, […]
Barcelona really, really, REALLY want Cesc Fabregas
July 10th, 2009
[Photo: PA] Firstly, let me say that I think Cesc Fabregas is very happy and settled at Arsenal. But that doesn't mean he's ruled out moving to Barcelona – his boyhood club – this summer. The two states of mind aren't mutually exclusive. The Cesc-to-Barca whispers are mounting, and it's not as if Barcelona president […]
Transfer Toss: Will Liverpool really bid for Franck Ribery?
July 10th, 2009
As this photo montage shows, Franck Ribery is back in training at Bayern Munich. But that hasn't stemmed the flow of transfer toss about Ribery's future. Most of Europe's major clubs have been linked with the wrong-faced Frenchman, and now you can add Liverpool to the list. This just in from Goal.com: "Bayern Munich winger […]
Barcelona 99% certain to sign David Villa
July 10th, 2009
So Nando Torres won't get David Villa as a playmate. Barcelona midfielder Xavi Hernandez has spilled the beans about the future of the Valencia striker. Xavi told Sport: "Talking to him [after the Confederations Cup], I noticed that he was very excited at the prospect of coming to Barcelona. "It is now only a matter […]
Sh*t Lookalikes: Fabricio Coloccini and Matt Bowman, lead singer of The Pigeon Detectives
July 10th, 2009
Horror Hair
Two very ugly men with wonky faces and indisputably shit hair. That is all. 'Nuff respect to Pies reader Kit Sheppard, who emailed us with this fine suggestion. Think you can do better than Kit? Email Pies with your lookalike(s).
A random football list: Top 10 shameful ponytails
July 10th, 2009
The ponytail is a particularly nasty hairstyle, rarely seen in real life these days, but it has a shamefully rich heritage amongst footballers… Brian Kilcline You're looking at a man's man's man's man. Even with his girly hair, you'd never, ever mess with Kilcline. Roberto Baggio The one and only Divine Ponytail. Gaze on it […]
Snapshot: Lionel Messi meets Mickey Mouse
July 10th, 2009
Lionel Messi's juggling skills impress the hell out of Mickey Mouse – what, hasn't Mickey ever been to SeaWorld? [Photo by Kent Phillips/Disney via ABACAUSA.COM] Messi was at the Florida resort yesterday to help promote the 'Disney Cup Youth International Soccer Tournament'. Mickey was there because, er, that's his job, to delight/scare small children.
Photo gallery: Llanelli 0-3 Motherwell, Europa League first qualifying round
July 10th, 2009
Europa League
Real, competitive football is here again! (And not a moment after the last season finished, it seems.) We ready to do this again? Phew… OK… Hell yes… Motherwell turned around a shock 1-0 first-leg deficit to reach the second qualifying round of the brand-new Europe League. They ran out comfortable winners against Llanelli at Parc […]
Snapshot Classic: Carlos Valderrama v Tab Ramos, USA '94
July 9th, 2009
International football
USA midfielder Tab Ramos attempts a sliding tackle on Colombia's Carlos Valderrama during a group game at the 1994 World Cup finals, held in the Rose Bowl in Pasadena. [Photo: PA] The US won the game 2-1, thanks to goals by Ernie Stewart and an infamous own goal by Andres Escobar, who paid for the […]
Photo Gallery: Chelsea unveil Daniel Sturridge
July 9th, 2009
'Pah! Yet another Englishman I 'ave never 'eard of.' New Chelsea signing Daniel Sturridge makes his first appearance in a Chelsea shirt. Solid signing by Chelsea. Again, not an Ancelotti signing a such but Sturridge looks an excellent prospect. I'm surprised Man City let him go. Click on the image below to launch the gallery…
Photo gallery: Liverpool unveil Glen Johnson
July 9th, 2009
G-Jo (that doesn't really work, does it?) swaps Pompey blue for Liverpool red. Photos taken today at Melwood. Click on image below to launch gallery. (Nice legs, Rafa. I just threw up in my own mouth.) [Photos: PA]


text:
Come find what you're looking for.
Available across all devices
Get premium!
Full access. No clicking. Hands-free navigation.
Monthly ($5.99) or Annual ($9.99)
All subscriptions are set to automatically renew. Visit your account's profile page to cancel your subscription. Sorry, but all sales are final.
"Long live the BUSH!"
- The_Naked_Me


text:
Here's how the No Man Left Behind model can attract new men to your church and to its ministry for men. read more
Charisma Magazine
Page 30 of 116
25 Things a Daughter Needs From Her Dad
Here are some honest, heartfelt things that girls—and grown women—are looking for from their fathers. read more
Here's a Great Opportunity to Show Christlike Love
We all know God hates divorce. But when it does rear its ugly head, shouldn't we carry out God's love to those involved? read more
4 Tips for Dating as a Single God's Man
The Bible doesn't give much information on dating, especially for this current culture. But here are some godly characteristics to aspire to. read more
The Most Important Thing a Man Must Remember About His Faith
Here's why your faith is not only a privilege; it's also a responsibility. read more
Why You Should Make Out With Your Spouse
If you're not normally given to public displays of affection—or to very little physical affection at all—here are some strong benefits simple kisses can bring. read more
Against the Odds, This Incredible Love Story Has Persevered For 75 Years
On the eve of Valentine's Day, Pastor Mark Rutland pays a heartfelt tribute to his parents, who recently celebrated their 75th wedding anniversary. read more
6 Ways to Blow a Special Day
With Valentine's Day upon us, how can you avoid these mistakes when it comes to your spouse? read more
5 Creative and Unique Types of Valentine's Gifts
It's an old saying, but it still holds true. It's not what you give, but what matters more is how much thought and care you put into a Valentine's Day gift. read more
Dad, Here Are Your Lines
If you're ready to kick it up a notch and daily invest in the heart space of your daughter, here is a decoder version of how girls understand and translate your responses to them. read more
15 Ways to Discern Jezebel's Assassins in Your Midst
In this season, the spirit of Jezebel is raging against believers and the church. This spirit seeks ...
Prophetic Dream: How to 'Buy' Sustaining Oil for Your Lamp
This is our season to buy oil, to purchase from the Lord that which is tried in the fire.
5 Truths to Understand When Leading Leaders
If you are in leadership for any length of time, there will probably come a time where you will need to lead other leaders.
What to Say (and Not to Say) to a Sexual Abuse Victim
Jesus brings good news to the abused, not shame, condemnation or put-downs.
7 Reasons Your Organization Suffers from Marketing Malpractice
Receive Dr. Steve Greene's 7 Reasons Your Organization Suffers from Marketing Malpractice, plus 3 Things to Do Now to Prevent Further Damage as your FREE download.
29 Protocols for Engaging in Deliverance Ministry
Casting out demons is not a science, but there are measures you should take to protect the person ...
Watch: John Paul Jackson Unmasks the Jezebel Spirit
In this interview on the 'Jim Bakker Show' just prior to his death in February 2015, John ...
2 Things You Don't Know About Yourself
Here are two reasons that you're a target for the enemy.
How to Respond When Your Faith Is Mocked
Christians weren't laughing when Saturday Night Live made fun of traditional values.
The Fourth Great Wave of the Holy Spirit Has Begun
Every previous wave of the Holy Spirit has restored truths to the larger body of Christ and often ...
Ruth Bell Graham's 5 Truths on Prodigals and Those Who Love Them
If you're praying for a prodigal, we hope you'll find wisdom and encouragement from the ...
When God Puts You Where You Don't Want to Be
Will you take the leap of faith in whatever it is He is calling you to do?
5 Truths to Understand When Leading Leaders
If you are in leadership for any length of time, there will probably come a time where you will need to lead other leaders.
5 Things You Need to Know to Find Your Calling
Here are some jewels of wisdom from a woman who has been in the ministry for over 40 years.
7 Reasons Your Organization Suffers from Marketing Malpractice
Receive Dr. Steve Greene's 7 Reasons Your Organization Suffers from Marketing Malpractice, plus 3 Things to Do Now to Prevent Further Damage as your FREE download.
Are You Aware of Your Blind Spots?
Others may notice, but sometimes we are not aware of them.
How to Walk in the Divine Promises of Health and Healing
What if speaking God's Word and praying its principles over your body acted as a healing ...
Should Christians Date?
Should a single Christian woman simply wait for God or actively pursue her future spouse?
How Worship Leader Darlene Zschech Walked Through Cancer
If you are walking through the valley of the shadow of death, all I can say to you today is hang on.
Bethel Beliefs on Signs, Wonders and Miracles
Miracles, in one form or another, have been a part of nearly every revival in church history.
7 Things to Do When You Are Frustrated With Life
What else do you do to overcome frustration?
God's Power at Work in the Church Today
Charisma's founder, Steve Strang writes about today's hottest topics affecting the Christian faith.
One of the Biggest Sources of Spiritual Delusion in the Church Today
Rick Joyner sounds off.
God created sex for the married man and woman. Shouldn't we treat it like the gift that it is?


text:
WANAQUE, N.J. (CBSNewYork/AP) — An elderly man who was found on fire outside his Passaic County home has died from his injuries.
But authorities say it's still not clear how the fire started.
Wanaque police told NJ.com that 71-year-old Robert Caywood had "severe burns" all over his body when officers found him at his Pellington Street home around 6:40 p.m. Friday. He was flown to a burn center, but a center spokeswoman said Sunday that Caywood had died.
Further details on his death were not disclosed.
No other injuries were reported in the fire, which remains under investigation by Wanaque police and the Passaic County Prosecutor's Office.
You May Also Be Interested In These Stories
- NYPD: Truck Driver Arrested After Fatal Bronx Crash With Bicyclist
- Moose Put Down After Being Struck By SUV In Connecticut
- Division Avenue High School Reopens After Asbestos Concerns
- Trump Says He Was 'Really Surprised' By Paul Ryan's Rebuff
(TM and © Copyright 2014 CBS Radio Inc. and its relevant subsidiaries. CBS RADIO and EYE Logo TM and Copyright 2014 CBS Broadcasting Inc. Used under license. All Rights Reserved. This material may not be published, broadcast, rewritten, or redistributed. The Associated Press contributed to this report.)


text:
This job posting is no longer available on Indeed.
Find similar jobs:Marketing Manager jobs
- Wild TV jobs
Wild TV is looking for a highly motivated Marketing Manager that has a passion for direct response marketing and maximizing profits while ensuring customer needs are met. Overseeing the marketing department, the selected candidate will be the point of contact for all internal and external communications including relaying management goals and objectives to the Wild TV marketing team and collaborate on campaign development and execution.
Job Description:
More information about this position and Wild TV is available on our website at www.wildtv.ca.


text:
- Product Information
Product DescriptionDress for the party in silver glitter. Our silver glitter fedora style hat will be a great choice for your black tie affair. If silver fits your color scheme for New Year's Eve, birthday or anniversary parties then our silver glitter fedora is a great choice to dress up for the party. Silver glitter fedoras are sold by the piece. Please order in increments of 1 piece.
- Questions & Answers
- Reviews
Customer Reviews
- Using them for my MS Walk Team- Perfect Review by Mefem2002
Love the hats. Bought silver (grey) hats and ties for my MS Walk team to wear at our Walk on May 5. Our team is 50 Shades of Grey Matter, and these props are perfect for a fun fundraising event day. Thank you so much. My friends like them so much, that more friends are walking with me, I may have to order more!!
(Posted on 2/28/13)
- Product satisfaction Review by Weze
I was really pleased with the products. It was more than I expected.
(Posted on 11/21/11)
- The hat was a perfect 'topper' Review by Party Guy
I bought 40 for a holiday party. They really helped get the actiion moving. I'm very happy with the product. It met my needs 110%
(Posted on 12/29/10)
Write Your Own Review
Brighten Your Cinco de Mayo Celebration with a Glow Fiesta!
With Cinco de Mayo coming up, you can spice up your fiesta by adding a glow in the dark theme! Mix neon colors with southwestern prints, toss in some glow, and you'll have yourself an unforgettable celebration!
Star Wars Theme Party Ideas
Celebrate Star Wars Day on May 4th with a Star Wars theme party. Put together an out-of-this-world celebration with the help of these party tips!
Host Your Own Kentucky Derby Party with These Simple Tips!
Join in on all the fun that the Kentucky Derby has to offer when you host a Kentucky Derby party in your own home! Wear your fancy hat and use these party ideas to prepare for your fabulous event.
You Recently Viewed
Los Angeles Lakers Lunch Napkins - 16 Pack
$4.40 Per 16 Pack
Blue LED Shot Glass-2oz
$1.90 Per piece
Black Glitter Top Hat
Night FootGolf Deluxe Kit
$109.95 Per Unit


text:
Invite Freelancer to Project
You don't seem to have an active project at the moment. Why not post a project now? It's free!Post a project
Auckland CBD, New Zealand
Member since November, 2012
0 Recommendations
siriuslydivine Online Offline
anything todo with forex or marketing is my territory...
$50 USD/hr
- N/AJobs Completed
- N/AOn Budget
- N/AOn Time
- N/ARepeat Hire Rate
Recent Reviews
No reviews.
- Facebook Connected —
- Preferred Freelancer —
- Payment Verified
- Phone Verified
- Identity Verified —
- Email Verified


text:
Does anyone supervise their children anymore? This was the worst experience I've ever had at a children's museum. The staff is very nice though.
This museum has great exhibits! Learn even more about prehistoric animals, and buy highly detailed dinosaur collectibles by visiting www.FindDinosaurs.com. Fine educational toys! Read more
Don't go on Wednesday it may be free but packed.
The toddler room is for toddlers, people. And where the hell are your parents?
Fun place for kids of alp ages.
Boring for adults but my kids love it
What a great place to volunteer!!!!


text:
Are you...?
If yes, click here to login.
The Pillbox Pharmacy
Phone: (808) 737-1777 Fax: (808) 737-4966 1133 11th Ave, Honolulu, HI 96816 ! BBB Business Review on The Pillbox Pharmacy is being Updated by BBB !
BBB Business Reviews may not be reproduced for sales or promotional purposes.
This company offers a drug store & pharmacy.
Reason for Rating
BBB rating is based on 13 factors. Get the details about the factors considered.
Based on BBB files, this business has a BBB Rating of No Rating. The reason is as follows:
- This business has no rating at this time because BBB file information is being reviewed and/or updated.


text:
Tell us if Jin You Yuan - Healthy Vegetarian is closed, moved, or if the info is incomplete or inaccurate.
To add a restaurant or store, use the: Add Form - OR - Add Photo | Write Review
Are you the Owner or Manager? Click here for more info regarding your listing


text:
Box Office Report: 'Transformers 4' Opens to $300 Million Worldwide
Paramount's 'Transformers: Age of Extinction' debuts to $100 million in North America and $201.3 million overseas, including a stunning $90 million opening in China.
Paramount's 'Transformers: Age of Extinction' debuts to $100 million in North America and $201.3 million overseas, including a stunning $90 million opening in China.
In a message taped for fans, Guillermo del Toro announces that Pacific Rim 2 is officially in the works alongside a continuation of the comic-book series 'Year Zero' and a new animated series.
Alfonso Cuaron's 'Gravity' is honored with five awards at the 40th installment of the Saturn Awards, hosted by the Academy of Science Fiction, Fantasy and Horror Films.
Marvel's 'Guardians of the Galaxy': An IMAX 3D First Look features exclusive footage to be shown in 150 select IMAX theatre locations across North America on July 7.
Based in Playa Vista, new facility offers 22,000 square feet, nine Red Epic cameras, QTake Video Assist System, four stereo rigs, extensive lens collection and DI projection theater.
The Academy of Motion Picture Arts and Sciences extends invitations to join the organization to 271 artists and executives who have distinguished themselves by their contributions to theatrical motion pictures.
Twentieth Century Fox releases a new behind-the-scenes featurette for 'Dawn of the Planet of the Apes' exploring the performance capture technology used by WETA Digital to transform actors into apes.
The Academy of Motion Picture Arts and Sciences updates regulations for how companies and individuals may market movies and achievements eligible for the 87th Academy Awards to Academy members.
500 Additional render farm management software licenses to be used on 'Teenage Mutant Ninja Turtles,' 'Transformers: Age of Extinction' and other blockbuster films.
Autodesk to acquire Shotgun Software, creator of cloud-based production management platform for film, television and games industries.
Prime Focus Limited announces the merger of its creative services subsidiary Prime Focus World and visual effects provider Double Negative; combined entity will be the world's largest integrated VFX, 3D Conversion & Animation services company.
A new trailer and four new character posters are released for the 'Teenage Mutant Ninja Turtles' live-action reboot directed by Jonathan Liebesman and produced by Michael Bay.
Beijing Pangu Investments, Ltd. and Paramount Pictures resolve the accusation that the studio breached its promise to feature the distinctive Pangu Plaza in the 'Transformers: Age of Extinction' film set to premiere Friday, June 27.
Blacklist Director Tendril teams with agency Ammirati NYC to create a colorful mix of CG and live-action for a new campaign for Seagram's Escapes.
Creative agency and production company Stardust creates a new international campaign featuring soccer superstar Neymar Jr. for China's wildly popular messaging app WeChat.
Technicolor-owned creative studio MPC collects a prestigious Gold Lion at Cannes for its work on the game trailer for Assassins Creed "Black Flag: Defy."
New AMD professional graphics card delivers GPU compute power for future-ready 4K workstation workflows, providing 38X more performance than closest competing product.
Disney's 'Maleficent' takes first place at the international box office with $44.7 million; DreamWorks Animation's 'How to Train Your Dragon 2' comes in second place with $43.5 million.
Less than a month ahead of the film's release, Fox unleashes the final trailer for 'Dawn of the Planet of the Apes.'
With Scott Derrickson set to direct, Marvel Studios negotiates with Jon Spaihts to write the script for 'Doctor Strange.'
Students at the for-profit California Art Institutes could get part of their education paid for under a $4.4 million settlement between the City of San Francisco and parent company Educational Management Corporation.
Led by VFX Supervisor Gary Brozenich and DFX Supervisor Matt Middleton, MPC delivers the finale sequence for 'Edge of Tomorrow.'
Nexus Director Kibwe Tavares is named one of the top 20 young global directors at Saachi & Saachi YDA showcase, receiving the prize for Best Short Film for 'Jonah' at the CFP-E and SHOTS YDA awards.


text:
M500 120GB SATA 6Gb/s Solid State Hard Drive - (CT120M500SSD1)
Read Speed: 500MB/Sec, Write Speed: 130MB/Sec, 62,000 IOPS, Controller: Marvell
Stock Code: HD-027-CR
Manufacturer: Crucial
Product information
"M500 120GB SATA 6Gb/s Solid State Hard Drive - (CT120M500SSD1)"Crucial M500 SATA 6Gb/s Solid State Drive - Successor to the M4!
Transform your system's performance.
New interfaces and multitasking demands require more from your system than perhaps ever before. From connecting instantly with friends and accessing apps, websites, and playlists online, to simultaneously streaming videos and downloading files, your computer is faced with an entirely new set of performance expectations that a hard drive struggles to meet. That's where the Crucial ® M500 SSD comes in. With data transfer speeds that are radically faster than a hard drive, the Crucial M500 SSD isn't just a storage upgrade - it's a complete system transformation. From its nearly instantaneous boot times, powerful data transfer speeds, increased multitasking capability, and rock- solid reliability, the Crucial M500 delivers dramatic performance gains – all at an affordable price.
- Interface: SATA 6Gb/s
- Sustained Sequential Read: 500MB/s
- Sustained Sequential Write: 130MB/s
- Random Read (4k transfer): 62,000 IOPS
- Random Write (4k transfer): 35,000 IOPS
- Form Factor: 2.5"
- NAND: 20nm Micron MLC
- Warranty: 3 year
- Controller: Marvell
- Endurance: 125TB total bytes written
More links for "M500 120GB SATA 6Gb/s Solid State Hard Drive - (CT120M500SSD1)"
Supplier description
Crucial - "The memory & storage experts". Whether it's a new operating system, new motherboard, or the latest ultra-high requirements game, Crucial is the first company to extensively test and optimise its products for maximum compatibility and performance. Over 35 years of technology experience can ensure top-tier quality across the board with not only their memory modules but also its growing range of SSDs and robust USB drives.
Customer review "Crucial M500 120GB SATA 6Gb/s Solid State Hard Drive - (CT120M500SSD1)"
1st SSD - happy!
Upgrade for my 2008 MacBook Pro which had mechanical hdd. Very happy with the performance gain.
I've read quite a bit about these drives and the 120GB and 240GB are said to have poor write speeds compared to peers. I don't need great writes for my Mac so price was my driver. Very happy with speed/price. Research says that if you need writes get a 480 or 960 - then the drives compete with peers.


text:
How can I wean my toddler from breastfeeding?
I'm 11 weeks pregnant and need to stop breastfeeding my toddler, who wakes up twice a night to be fed. I've tried feeding her right before she goes to sleep, not going to her in the night, giving her cows' milk instead of breast milk, and having her father comfort her -- but nothing works. She tries to lift my shirt and nurse. Please help.
Posted: 04/24/2002 by a BabyCenter Member
Mom Answers
Sort by: best answers | most recent answers
251 - 260 of 305 answers
The World Health Organisation recommends breast feeding for 2 years. This is quite standard in most parts of the world. Those who find it distasteful to breastfeed beyond a few month are entiltld to their own opinions but to suggest that a natural and normal act is in some way perverse says more about their own psyches than anything else.posted 08/24/2007 by Sophia100
Whatever you decide to do, do it gradually, with loving respect for your child's feelings. Putting something foul tasting on your breast is a disgusting horrific idea, don't do that.posted 02/24/2004 by a BabyCenter Member
The World Health Organisation recommends breast feeding for 2 years. This is quite standard in most parts of the world. Those who find it distasteful to breastfeed beyond a few month are entiltld to their own opinions but to suggest that a natural and normal act is in some way perverse says more about their own psyches than anything else.posted 08/24/2007 by Sophia100
There is absolutely no reason why your toddler needs to be fed at night. You're just prolonging weaning because it makes YOU feel better. Are you going to get up at night and nurse both of them? Have your husband go in at night, comfort the toddler, and tell her it's time to be a big girl and go back to sleep.posted 03/03/2013 by a BabyCenter Member
I desperately want to wean my 13-month old off the breast but he never took a bottle & does not drink from a sippy cup, straw or regular cup. What can I do?posted 07/10/2006 by newmom0606
I have a 17 month old little girl (17 today) and I am still breastfeeding. Last time we visited the Dr. She say there was no real reason for me to still be breastfeeding. I told her I only breastfeed during the night. (we sleep together and I don't know if her being that close to her milk is not helping to wean her off). She just started Daycare and sometimes when I go see her she has the need for nursing so I nurse her. It seems to make her happy. I don't mind breastfeeding and I know I will have to wean her sooner or later. My Doctor said that I was going to cause her tooth decay by breastfeeding her during the night.... is this possible? I know I have to quit but I just cannot just cut her off. I feel that would be cruel, but I am jeopardizing her dental healh then that's different. How right is this Dr. ? She pretty much made it sound like there was no nutritional value in the breastmilk at this age..posted 03/31/2005 by a BabyCenter Member
I am still nursing my 19 month old daughter but have heard of putting oil of clove on your nipples. It might smell a little but will not sting like the thumz stuff.posted 04/21/2005 by punkinbuttr
I have tried `anonymous's suggestion of using Thum. It worked. My toddler sucked twice, and never asked for the breasts again. (I have tried so many things, even wasabi, the Japanese relish.) But I have to warn you that it is not easy to wean a toddler. My daughter didn't know how to go to sleep without sucking and was crying and screaming for almost a week. I started with the day feeds then the night feeds. Took me 1 month to totally wean off my toddler. It was painful.posted 06/26/2005 by christabel_lee
Moms who won't give their children what's best, both physically and emotionally, are the ones who make ME sick. The AAP recommends at least one year of breastfeeding. Women who choose to go longer are only extending the many benefits to their children. How can anyone criticize that?? Most literature says it's best to let your child decide when. If you can't, then do it gradually and replace it with the equivalent level of warmth and attention. Good luck!posted 02/22/2004 by a BabyCenter Member
[There is no way I personally would keep breastfeeding past a year old. But thats just my opinion. It's completely ridiculous to keep feeding from the breast when the child is able to feed him/herself. It shows laziness.] It is actually recommended for breast feeding to continue until the age of two. It has been stated over and over again that breast feeding is beneficial as long as it continues. Breast feeding is a choice mothers make who want the best for their child's emotional and physical well being, it is not laziness. Laziness is sitting or laying a baby down somewhere and giving him or her a bottle to feed to themselves. When you nurse your child, you take time out of your day to give him or her your undivided attention. This actually involves more time than letting the baby feed itself a bottle. My son eats table food and is even starting to use a spoon, however, he still enjoys the bond and one on one time and attention that nursing gives us. Did you breastfeed?posted 11/28/2007 by headernoel
Answer this question


text:
Too Much Love celebrates 5 years on Saturday
Photo by Dave Eckblad
It's hard to imagine a weekend at First Avenue without Too Much Love at this point. Soviet Panda has dedicated hundreds of Saturday nights to the young and the restless of Minneapolis since the event's incarnation in 2007. That's five years, folks. And in dance night years, it's a lifetime.
Soviet Panda, a.k.a. Peter Lansky, says there might be a few little surprises up his sleeve to commemorate the occasion, but the night will take shape with the crowd and their energy.
"The party is mostly about no gimmicks," Lansky says. "We care about music, dancing and drinking."
The music in this case will be provided by local techno wunderkind Dustin Zahn, whose productions have taken him all around the globe -- most recently to Berlin, where Lansky was visiting, and the two were able to hang out.
"I'm always excited to hear Dustin play," Lansky says. "His sets really blow me away."
And celebrating to good weather would be an added plus: "I think the night will go well, though I hope it doesn't rain again, that has been hurting us these past couple weeks."
Looking back, Lansky says five years of TML has really been a huge learning experience, as the night was birthed from "a kind of musical consensus" about what was missing from Minneapolis clubs thanks to "the current mainstreaming of electronic music."
Owing a $1 admission for students, a good portion of the Twin Cities college-kid contingency owes many a night to Lansky and his featured DJs, which over the years have included LCD Sounsystem, Gigamesh, DVS1 and many other noteables. (The party, for the record, is sponsored by this very paper).
So, this Saturday, don your most colorful clubby ensemble, take a few pics in the Photobooth for us, and burn up the dancefloor in celebration of one of Minneapolis's defining nightlife events.
TOO MUCH LOVE with Soviet Panda & Dustin Zahn takes place on Saturday, May 5. Admission is $3/$1 with college I.D. 18+. 10 p.m.
Photo by Dave Eckblad
Get the Music Newsletter
Keep your thumb on the local music scene each week with music news, trends, artist interviews and concert listings. We'll also send you special ticket offers and music deals.
In Case You Missed It
Popular Stories
Sponsor Content


text:
Becoming a Manager: EBook EditionThe role of the new manager demands a new mindset, new activities, and new relationships with people throughout the organization. Becoming a Manager guides the first-time manager through these and other challenges. Part One, Making the Transition, explores how to make the critical shift from individual contributor to manager; what it takes to build a successful partnership with your boss; and the
The Busy Manager's Guide to DelegationDelegation amounts to a lot more than just passing work off onto subordinates. When handled correctly, it gives managers a chance to strengthen their departments by developing the skills and organizational competencies of their people. Filled with quick tips, exercises, self-assessments, and practical worksheets, this book presents an easy-to-master five-step process for effective delegation.
Asserting Yourself At Work: EBook EditionTechniques for managing others while maintaining mutual respect. Asserting Yourself at Work provides business professionals with the communication tools and psychological foundation they need to perform more assertively on the job. Designed for front-line managers, supervisors, team leaders, team members, employees, and life-long learners, this course promotes the use of direct, inclusive
Best Practice Workplace Negotiations: EBook EditionBest Practice Workplace Negotiations offers a systematic approach to developing negotiating skills. It serves as an introduction to current best practices in negotiation that can be applied across a broad range of business situations. This up-to-the-minute course covers win-win vs. win-lose negotiations; the BATNA concept (best alternative to a negotiated agreement—what every negotiator should
Interpersonal Communication Skills in the Workplace: EBook EditionEffective communication is an important element of success for every organization, leader, manager, supervisor, and employee. Good communication skills are a prerequisite for advancement in most fields and are key to exercising influence both within and beyond the work group. This edition retains the subject matter strengths of the previous version and augments them with content that reflects new
HBR Guide to Managing Up and AcrossARE YOUR WORKING RELATIONSHIPS WORKING AGAINST YOU?To achieve your goals and get ahead, you need to rally people behind you and your ideas. But how do you do that when you lack formal authority? Or when you have a boss who gets in your way? Or when you're juggling others' needs at the expense of your own?By managing up, down, and across the organization. Your success depends on it, whether you're
Over 950 Practical Lessons Your MBA Professor Didn't Teach YouIt's not that Cy Charney opposes MBA and other types of academic training. It's just that he feels academic training doesn't go far enough -- that it fails to prepare people for what life as a manager is really like.Now Charney has published the ninth book in his lifelong campaign to bridge this gap -- a gap that he believes is particularly challenging in today's instant, multi-tasking, change
Leadership Behavior Impact on Employee's Loyalty, Engagement and Organizational Performance
Leadership Behavior and Employee Perception of the OrganizationEvery organization is looking for ways to improve employee participation, loyalty and engagement; which most scholars believe could <SPAN style="FONT-SIZE: 11pt; FONT-FAMILY: 'Adobe Garamond Pro'; mso-bidi-font-family: Calibri; mso-fareast-font-family: 'Times New ...
Teambuilding That Gets Results: Essential Plans and Activities for Creating Effective TeamsTeambuilding is one of the fastest-growing areas on the business shelf, as companies look to increase efficiencies and morale. Teambuilding That Gets Results, an easy-to-use guidebook for building effective teams, will increase the strength of any business. Harriet and Linda Diamond offer expert advice gleaned from years of consulting businesses on building strong teams. Topics include: --Building
$13.99In today's competitive workplace, your ability to communicate is your most important business skill.This valuable handbook to better business communication can help you develop the skills you need to succeed. Using real-life examples, it offers practical, easy-to-use instruction in writing effective memos and reports, making memorable presentations, and leading productive meetings. It also
The Everything Career Tests Book: 10 Tests to Determine the Right Occupation for YouAmerican workers are upwardly mobile movers and shakers who change careers often, always on the search for their perfect niche. But you can't follow your bliss unless you know what your bliss is. Enter The Everything Career Tests Book-your key to determining the career path you were destined for! This engaging, accessible guide boasts ten different tests that reveal the work habits, affinities,
The Practice of Leadership Coaching from the World's Greatest Coaches
J-B US non-Franchise Leadership (Book #152)When it was published in 2000, Coaching for Leadership became an instant classic in the field of executive coaching. This second edition updates and expands on the original book and brings together the best executive coaches who offer a basic understanding of how coaching works, why it works, and how leaders can make the best use of the coaching process. This thoroughly revised edition reflects


text:
We regret that due to the technical limitations of our site, we are unable to offer eBooks or Audio Downloads to customers outside of the UK.
For further details please read our eBooks help.
Software Engineering Education & Training, 11th Conference (Csee '98) (Hardback)IEEE
Hardback Published: 30/04/1998
- Not in our warehouse
Papers proceeding from the 11th Software Engineering and Training Conference, held in 1998. The volume is aimed at researchers, practitioners and students in software design and development.
Publisher: IEEE Computer Society Press,U.S.
You may also be interested in...
ReviewsWrite your review
We would love to hear what you think of Waterstones. Why not review Waterstones on Trustpilot?


text:
|Home | About | Journals | Submit | Contact Us | Français
Proteins perform most cellular functions in macromolecular complexes. The same protein often participates in different complexes to exhibit diverse functionality. Current ensemble approaches of identifying cellular protein interactions cannot reveal physiological permutations of these interactions. Here, we describe a single molecule pull-down (SiMPull) assay that combines the principles of conventional pull-down assay with single molecule fluorescence microscopy and enables direct visualization of individual cellular protein complexes. SiMPull can reveal how many proteins and of which kinds are present in the in vivo complex, as we show using protein kinase A. We then demonstrate a wide applicability to various signaling proteins found in cytosol, membrane, and cellular organelles, and to endogenous protein complexes from animal tissue extracts. The pulled down proteins are functional and are used, without further processing, for single molecule biochemical studies. SiMPull should provide a rapid, sensitive and robust platform for analyzing protein assemblies in biological pathways.
Dynamic interactions between proteins guide almost every aspect of cellular function1. Understanding how the macromolecules in living cells interact, holds the key to deciphering their roles in cellular function and regulation2, 3. Individual proteins are also part of diverse sets of protein networks, making it very challenging to tease apart various permutations of protein-protein interactions occurring in the cellular context4. Currently, the gold standard for determining interactions between proteins is the co-immunoprecipitation assay5–7 which relies on affinity-based co-purification of interacting proteins, followed by identification via Western blot (WB) or mass spectrometry. It is however difficult to determine how many copies of which proteins are present in the physiological complex using the conventional immunoprecipitation. In addition, many hours and multiple steps that often exist between sample preparation and measurements present uncertainties over the extent to which in vivo interactions are preserved prior to analysis.
In situ imaging methods based on resonance energy transfer8, 9, fluorescence correlation spectroscopy10, 11, two-hybrid methods12, 13 and bimolecular fluorescence complementation assay14 are other popular tools for studying pair-wise protein interactions. However, these methods cannot be applied to endogenous proteins and are, in general, blind to heterogeneous interactions between proteins and their stoichiometry.
Here we present a simple, direct and sensitive method to study cellular protein complexes with single complex resolution. We call this method single molecule pull-down or SiMPull because physiological macromolecular complexes are pulled down from cell or tissue extracts directly to the imaging surface of single molecule fluorescence microscopy.
The key requirement for pull-down assays is the selective capture of a protein of interest (bait), which will bring along its binding partners (prey). We constructed a flow chamber using a microscope slide and a cover slip, passivated with mPEG (methoxy polyethylene glycol)15 to prevent non-specific adsorption of cell extracts and antibodies, which should minimize false positives7. The imaging surface was also doped with biotinylated PEG and streptavidin, followed by biotinylated antibodies against the bait protein (Fig. 1a–d, Supplementary Fig. 1). When cell extracts are infused in the flow chamber, the surface tethered antibody captures the bait protein together with any interacting partners. After washing away the unbound cell extract, co-immunoprecipitated prey proteins are visualized either through immunofluorescence labeling (Fig. 1a) or using genetically encoded fluorescent protein tags (Fig. 1b). This approach is extendable to multi-protein complexes via multi-color labeling and has the potential to differentiate between multiple sub-complexes and configurations (Fig. 1c). When proteins are fluorescently labeled with a fixed ratio, photobleaching events yield stoichiometric information16, 17 (Fig. 1d).
We first validated the SiMPull assay for specific pull-down of yellow fluorescent protein (YFP) from cell extracts. When the crude lysate from cells over-expressing (His)6-tagged YFP was infused into the flow chamber coated with anti-His antibody, we observed single YFP molecules (Fig. 1e, f), similar to the analysis performed using purified protein18 (Supplementary Fig. 2). Binding of YFP to the antibody was stable over two hours (Supplementary Fig. 3). The blank slide surface showed ~30 fluorescent spots per imaging area, 2,500 µm2, possibly due to surface impurities. The number of fluorescent spots per imaging area, Nf, due to specifically pulled down proteins was 10–20 fold over the background; we could maintain > 10 fold signal to noise ratio by controlling the lysate dilution factor. Control experiments with YFP lysate on non-specific antibody and lysate without YFP expression showed the same Nf as blank. Even lysate with 10-fold higher concentration of YFP yielded only ~30 additional spots relative to the blank, implying less than 0.5% contribution from non-specifically adsorbed proteins (Fig. 1e, f). Nf increased monotonically as the cell lysate concentration is increased over three orders of magnitude (Supplementary Fig.4), showing that SiMPull can provide a quantitative estimate of protein concentration in cell lysate.
Single molecule photobleaching analysis performed using the monomeric YFP and tandem dimeric YFP constructs (Supplementary Fig. 5, 6) showed that accurate stoichiometric information can be obtained from the pulled down proteins when we account for the fact that about 75% of YFP is fluorescently active16.
Next, we demonstrate the ability to pull-down single protein complexes from cell extracts using cyclic adenosine monophosphate (cAMP) dependent protein kinase, PKA, as our model system. PKA is a ubiquitous serine/threonine kinase that acts downstream of the G-protein coupled receptor (GPCR) pathway19. In the inactive state, PKA is a tetrameric complex consisting of two regulatory (R) and two catalytic (C) subunits. In the presence of cAMP, the complex dissociates, thereby activating the enzyme. We prepared C-HA-YFP and R-Flag-mCherry constructs (Fig. 2a).
When only C-HA-YFP was expressed in HEK293 cells, we could specifically pull-down the protein from the cell lysate using surface immobilized antibodies against HA- or YFP-epitope (Fig. 2b). As expected, these samples did not show any detectable fluorescence above background in the mCherry detection channel (Supplementary Fig. 7).
When the two subunits, R and C, were co-expressed, WB showed that R and C coimmunoprecipitate20 and they dissociate when cAMP is added to the lysate, confirming that the modified constructs retain the known properties of PKA (Fig. 2a). In a similar fashion, when we pulled down R-Flag-mCherry with anti-Flag antibody, we could detect both YFP and mCherry fluorescence spots. The number of fluorescent spots in mCherry and YFP channels was similar, indicating, on average, a one-to-one association (Fig. 2c, d). 57% of YFP spots colocalized with a corresponding mCherry, as shown by individual images and their overlay (Fig. 2d). Incomplete colocalization may arise from basal tonic activation of PKA, inactive chromophores16, 21 or unbalanced expression of two proteins in individual cells. Adding cAMP analog to the flow channel or pre-incubating the lysate with cAMP resulted in a greatly reduced number of C subunit (YFP spots) without a significant change in R subunit (mCherry spots) (Fig. 2c, e). After the reaction, only 4% of remaining YFP molecules colocalized with a corresponding mCherry.
Intracellular levels of cAMP can be increased by stimulating GPCRs for activation of adenylyl cyclases. When the cells over-expressing the PKA complex were stimulated with forskolin, an agonist for adenylyl cyclase for cAMP production, the amount of active PKA in cells increased as seen via dissociation of PKA in WB (Supplementary Fig. 8). Similarly, in our SiMPull assay, the number of C subunit (YFP molecules) pulled down through R subunit decreased significantly (Fig. 2c).
We explored the stoichiometry of PKA via photobleaching analysis. When C-HA-YFP was expressed alone and pulled down using HA-antibody, 91% of YFP traces exhibited one-step photobleaching indicating a monomeric population (Fig. 2f). When C-HA-YFP and R-Flag-mCherry were co-expressed and pulled down using anti-Flag antibody, 47% of YFP traces displayed two photobleaching steps (Fig. 2g) while 51% molecules bleached in one step. Assuming a 75% active fraction of YFP16, this is consistent with the known stoichiometry of two catalytic subunits in each PKA. We did not perform photobleaching-based stoichiometry analysis for mCherry due to its inferior photophysical properties22.
Next, we examined the applicability of SiMPull to protein complexes from various cellular environments using different capture and detection configurations (Fig. 3).
Membrane protein complexes are particularly difficult to analyze using conventional methods, thus motivating new approaches23, 24. Their stoichiometry cannot be determined via photobleaching in the cell unless the areal density of protein complexes is low enough for single molecule detection16, 25, 26. SiMPull should be able to detect individual complexes if membrane patches containing one complex can be isolated. As a test, we applied SiMPull on β2 adrenergic receptor (β2AR), a prototypical GPCR. HEK293 cells were transfected with Flag-YFP-β2AR and membrane proteins were solubilized. We could specifically pull-down the receptor using antibodies against YFP or Flag (Fig. 3a–c). 29% of the traces displayed two distinct bleaching steps (Supplementary Fig. 9a), indicating a ~51% dimer population, assuming 75% of active fluorophores. Less than 3% of the traces showed 3 or more photobleaching steps. Our observation of β2AR homo-dimerization is consistent with previous studies27, 28. To test if β1AR may form heterooligomers with β2AR28, we co-expressed mCherry-β1AR and YFP-β2AR. Using antibody against mCherry-β1AR, we could pull-down YFP-β2AR and vice versa (Supplementary Fig. 9b–e). The two fluorophores colocalized with ~42% overlap, consistent with heterooligomer formation.
MAVS (mitochondrial antiviral signaling) is a mitochondrial outer membrane protein involved in innate immune response29. When isolated mitochondrial fraction from cells over-expressing YFP-MAVS30 was applied to a surface with anti-YFP, we observed bright fluorescent structures (Fig. 3e, left), indicating the presence of multiple YFP molecules. Upon pre-solubilizing the mitochondrial preparation using mild detergent, we observed isolated single YFP spots (Fig. 3e, center), 86% of which displayed single photobleaching steps (Supplementary Fig. 10), supporting monomeric state of solubilized MAVS. This observation suggests that the bright fluorescent structures observed in unsolubilized preparations are due to immunoprecipitated mitochondrial membrane patches or whole mitochondria. It may be possible to specifically immobilize cellular organelles or their components using antibodies against suitable marker proteins and perform single molecule measurements in a physiologically relevant context.
We extended the assay to detection via antibodies using mammalian target of rapamycin complex 1 (mTORC1) as a model system. mTORC1 is a key signaling complex that regulates cell growth and metabolism in response to nutrient availability to the cells31, 32. In addition to mTOR (mammalian target of rapamycin), a defining component of mTORC1 is Raptor (regulatory associated protein of mTOR), which associates with mTOR at an equimolar ratio31, 33. We expressed Flag-mTOR and HA-Raptor in HEK293 cells. Flag-mTOR was pulled down using biotinylated Flag-antibody; Raptor was detected using HA-antibody followed by fluorescently labeled secondary antibody (Fig. 3g). When both Flag-mTOR and HA-Raptor were co-expressed, we observed the detection antibody binding as fluorescent spots whereas background level of fluorescence was detected when only one of the two proteins was expressed (Fig. 3h, i), demonstrating antibody-based detection in SiMPull.
Exogenous expression may lead to non-physiological associations between proteins. Pull-down of endogenously expressed proteins, though desirable, is challenging due to low abundance, high background interaction with other cellular proteins, and general lack of high affinity antibodies. We tested if SiMPull can be used to detect interactions between endogenous proteins. A kinase anchoring proteins (AKAPs) bind to PKA and confine it to discrete locations in the cell34. Fig. 3k shows AKAP150 can be co-immunoprecipitated with PKA from mouse brain extract.
Primary antibodies against proteins are often expensive and difficult to label with biotin or fluorophores. Thus, to keep our approach general, we used biotin labeled secondary antibody to immobilize the antibody against the bait (PKA), and applied mouse brain extract. On probing for the prey protein (AKAP150) using its primary antibody and fluorescently labeled secondary antibody, we observed 10-fold more fluorescent spots in the channel with PKA antibody as compared to the control channel (Fig. 3l, m). SiMPull required a 20-fold lower sample volume as compared to the corresponding WB. This sensitivity allowed detection of PKA-AKAP binding from mouse heart tissue, which was below the detection limit of the conventional WB under the same conditions (Supplementary Fig. 11).
A key advantage of SiMPull is that protein complexes can be directly observed from a fresh cell lysate, bypassing purification procedures. We tested if SiMPull can be used for functional analysis of pulled down proteins. PcrA, a superfamily 1 helicase, is an ATP driven motor protein that binds and translocates on single stranded DNA (ssDNA)35. (His)6-tagged PcrA was pulled down from bacterial lysate using anti-His antibody and fluorescently labeled DNA molecules were added to the flow channel (Fig. 4a). Fluorescent spots due to labeled DNA binding appeared in the flow channel with pulled down PcrA, while the control channel showed minimal DNA binding (Fig. 4b, c).
When PcrA binds to a partial duplex DNA with a 5' overhang, it anchors itself to the junction and repetitively reels-in the ssDNA35. By labeling the DNA with a donor at the tail end and an acceptor at the junction (Fig. 4a), we could observe the reeling-in activity as a gradual increase in fluorescence resonance energy transfer (FRET) (Fig. 4d). Once PcrA reaches the end of ssDNA, it runs off the ssDNA track and repeats the process from the junction over and over, resulting in cyclic increases and decreases in FRET. 86% (161 of 188) of bound FRET-labeled DNA molecules exhibited repetitive cycling. On increasing the ATP concentration, translocation became faster (Supplementary Fig. 12a), and in the absence of ATP, DNA remained bound but no reeling-in activity was observed (Supplementary Fig. 12b). The mean translocation time matched well with the data obtained with purified protein (Fig. 4e, f). Thus, SiMPull can pull-down functional macromolecules directly from cell extracts for subsequent single molecule biochemistry in situ.
We have established a single molecule platform for analyzing cellular association of macromolecules. SiMPull can be used as an extension of commonly used WB analysis without requiring additional sample preparation (Supplementary Fig. 13) and confers several key advantages. 1) It can provide quantitative data on sub-populations of different association states. 2) It provides information on complex stoichiometry if the proteins can be stoichiometrically labeled. 3) The high sensitivity allows the studies of complexes of low abundance and a suitable calibration (Supplementary Fig. 4) should make it possible to determine the expression level of protein complexes in cell lysate. 4) The whole assay took about 30 minutes, considerably shorter than conventional WB. In a pilot experiment, we could dilute the cell lysate, pull-down and quantify YFP in 2.5 min (Supplementary Fig. 14). Therefore, it should be possible to analyze even relatively weak protein complexes as long as the dissociation rate constant koff is equal to or smaller than 0.01 s−1. Combining this with microfluidics platform, cross-linking methods or zero mode waveguide36 may extend the method to complexes with even higher koff.
Cellular processes are under a tight spatio-temporal regulation. In order to overcome ensemble averaging over heterogeneous cell populations, SiMPull may be combined with fluorescence aided cell sorting to selectively analyze a subpopulation or may even be pushed to the single cell level. In a preliminary experiment, we could pull-down and quantify proteins from 10 cells obtained through cell sorting (Supplementary Fig. 15) compared to ~5000 cells usually required for a WB37. Single cell SiMPull may enhance the recently demonstrated ability to quantify proteins and RNA numbers in single cells38. As in conventional WB, the sensitivity and specificity of SiMPull are determined by the quality of capture and detection antibodies. The assay may be combined with recent developments in labeling strategies39 for further improvement in sensitivity and labeling efficiency.
Post-translational modifications play an important role in cellular processes but are difficult to reproduce in recombinant proteins. In addition, the necessary co-factors or ligands for a protein of interest are often unknown. SiMPull as a preparatory tool provides a possibility to study these modified proteins or protein complexes that cannot be purified using conventional methods.
Flow chambers were prepared on mPEG passivated quartz slides doped with biotin PEG15. Biotinylated antibodies were immobilized by incubating ~10 nM of antibody for 10 min on NeutrAvidin (Thermo) coated flow chambers. Prism type total internal reflection fluorescence (TIRF) microscope was used to acquire the single molecule data40. Samples with fluorescent protein tag were serially diluted to obtain well-isolated spots on the surface upon 20 min of incubation over immobilized antibody surface. All dilutions were made immediately before addition to the flow chamber in 10 mM Tris-HCl pH 8.0, 50 mM NaCl buffer with 0.1 mg/ml bovine serum albumin (New England Biolabs), unless specified. Unbound antibodies and sample were removed from the channel by washing with buffer twice between successive additions. For immunofluorescence detection, immunoprecipitated complexes were incubated with a different antibody against prey protein (~10 nM) for 20 min and fluorescent-dye-labeled secondary antibody (2–5 nM) for 5 min before imaging. Single molecule analysis was performed using scripts written in Matlab.
Flow chambers were prepared on mPEG passivated quartz slides doped with biotin PEG15. Biotinylated antibodies were immobilized by incubating ~10 nM of antibody for 10 min on NeutrAvidin (Thermo) coated flow chambers. Prism type total internal reflection fluorescence (TIRF) microscope was used to acquire the single molecule data40. Samples with fluorescent protein tag were serially diluted to obtain well-isolated spots on the surface upon 20 min of incubation over immobilized antibody surface. All dilutions were made immediately before addition to the flow chamber in 10 mM Tris-HCl pH 8.0, 50 mM NaCl buffer with 0.1 mg/ml bovine serum albumin (New England Biolabs), unless specified. Unbound antibodies and sample were removed from the channel by washing with buffer twice between successive additions. For immunofluorescence detection, immunoprecipitated complexes were incubated with a different antibody against prey protein (~10 nM) for 20 min and fluorescent-dye-labeled secondary antibody (2–5 nM) for 5 min before imaging. Single molecule analysis was performed using scripts written in Matlab.
Prism type TIRF microscope was used to acquire single molecule data40. YFP was excited at 488 nm; mCherry was excited at 532 or 568 nm. Narrow band-pass filters were used to avoid cross-talk between channels (HQ 535/30 from Chroma Technology for YFP and BL 607/36 from Semrock Inc. for mCherry). All experiments were performed at room temperature (22 – 25 °C) unless specified. Single molecule analysis was performed as described earlier15. Mean spot count per image (imaging area 2500 µm2) and standard deviation were calculated from images taken from 20 or more different regions.
Single molecule fluorescence time traces of surface immobilized YFP-tagged proteins were manually scored for the number of bleaching steps16. To avoid false colocalization, samples were immobilized at an optimal surface density (~300 molecules in 2500 µm2 imaging area). The number of photobleaching steps (single frame intensity drops of equal size) in each trace was manually determined, following published procedures16. Fluorescence trace of each molecule was classified as having 1–4 bleaching steps or was discarded if no clean bleaching steps could be identified (Supplementary Fig. 5). Some fluorescent protein molecules exhibited blinking but under most circumstances, distinct fluorescence intensity levels could still be readily determined in spite of blinking (Supplementary Fig. 5a, b). Separate counts were maintained for each case. At least 500 molecules were analyzed for each sample. The probability of missed bleaching events, due to simultaneous bleaching of both FPs within the same imaging window is ~5%. The population distribution of observed bleaching events and discarded traces is reported in Supplementary Table 1. For future extensions to complexes with many more copies of the same protein, automated algorithms for scoring photobleaching steps would be required41.
Co-localization between YFP and mCherry was performed using a method similar to Ulbrich et al.42. Briefly, we took two separate movies of the same region using YFP and mCherry excitation. The fluorescent spots in both images were fit with Gaussian profiles to determine the center positions of molecules to half-pixel accuracy. Next, for each molecule in YFP image, we determined the mCherry molecules with center within 2 pixel (~300 nm) distance. The number of molecules where this co-localization occurred divided by the total number of YFP molecules was presented as overlap %.
As YFP has been shown to dimerize, monomeric YFP was generated through site directed mutagenesis of alanine 207 to lysine using pEYFP-C1 as DNA template (Clontech). For bacterial expression, monomeric YFP was cloned into Sal I and Xho I sites of pET-28b(+) vector. BL21 DE3 cells were transformed with YFP construct and induced by 0.2 mM IPTG for protein expression. Cells were resuspended in lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole pH 8.0) and sonicated. The lysate was centrifuged at 15,000g for 20 min to collect supernatant used for SiMPull.
For expression in mammalian cells, YFP-(His)6 was generated through addition of a 6xHis-tag to the C-terminal of YFP and subcloned into Xho I and Xba I sites of pCDNA3.1(+). A second YFP was subcloned into the Hind III and EcoR I sites of pCDNA3.1-YFP-(His)6 to make a tandem dimeric YFP construct. Monomeric and dimeric YFP constructs were transiently expressed in HEK293 cells and purified using standard Ni-NTA chromatography. Proteins were detected by WB using GFP antibody (Clontech) or Penta-His antibody (Qiagen). For single molecule analysis, samples were immobilized on biotinylated anti-Penta-His antibody (Qiagen) or on biotinylated polyclonal anti-GFP antibody (Rockland Immunochemicals, Inc.).
HEK293 cells were transfected with R-Flag-mCherry and C-HA-YFP constructs. The regulatory subunit used was PKA RIIβ, and the catalytic subunit is the Cα isoform. After 24 hr expression, cells were harvested into lysis buffer (10 mM Tris pH 7.5, 1% NP-40, 150 mM NaCl, 1 mM EDTA, 1 mM benzamidine, 10 µg/ml leupeptin, 1 mM NaF, 1 mM Na3VO4). This lysate was centrifuged at 14,000g for 20 min and used for SiMPull. For bulk immunoprecipitation, anti-Flag M2 beads were added to the lysate for 3 hr at 4 °C. Proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes for blot with anti-HA antibody and anti-mCherry antibody.
cAMP treatment: A non-hydrolysable analog (8-Br-cAMP, Sigma), was used to activate PKA. For in vivo stimulation in cells, R-Flag-mCherry and C-HA-YFP were transiently expressed in HEK293 cells for 24 hr. Cells were pre-treated for 10 min with 10 µM 3-Isobutyl-1-methlxanthine (IBMX, Sigma) and 5 min stimulation with 10 µM Forskolin (Sigma). Cells were immediately washed with cold PBS and lysed as described earlier.
Flag-YFP-β2AR, HA-YFP-β2AR, Flag-mCherry-β1AR and HA-mCherry-β1AR were transiently expressed in HEK293 cells for 24 hr. Cells were harvested into hypotonic lysis buffer (10 mM Tris pH 7.4, 1 mM EDTA, 1 mM benzamidine, 10 µg/ml leupeptin, 0.3% DDM (n-dodecyl-beta-D-maltoside), and incubated for 30 min before centrifugation at 600g for 10 min. Supernatants were collected and used for SiMPull with antibodies as indicated.
HEK293 cells were transiently transfected with YFP-MAVS30. Intact mitochondria were isolated using MITOISO2 kit (Sigma) and diluted in the storage buffer supplied with the kit. Mitochondrial preparation was immobilized on slides either directly or after solubilization by adding 1% DDM to the storage buffer. We obtained similar results using whole cell lysates prepared using several different lysing solutions. YFP-MAVS or mitochondria were immunoprecipitated using biotinylated antibody against GFP.
Flag-mTOR was stably transfected in HEK293 cells to obtain near endogenous expression levels of mTOR. For mTORC1 pull-down, Flag-mTOR stable cell lines were transiently transfected with HA-Raptor. HA-Raptor only lysate was obtained by transiently transfecting HEK293 cells with HA-Raptor. Cells were lysed using CHAPS detergent buffer (40 mM HEPES pH 7.5, 0.3% CHAPS, 150 mM NaCl, 2.5 mM sodium pyrophosphate, 1 mM β-glycerophosphate, 1 mM EDTA), with protease inhibitor cocktail. Lysate was diluted in the buffer without CHAPS for SiMPull with biotinylated anti-Flag antibody (Sigma). Co-immunoprecipitated HA-Raptor was detected using Goat-anti-HA antibody (Genscript) and Donkey-anti-Goat secondary antibody (Rockland Immunochemicals, Inc.) labeled with Cy3.
Mouse brain and heart extracts were prepared from two-week-old FVB mice after anesthetization. Both sample were homogenized in lysis buffer (20 mM HEPES pH 7.4, 0.5% Triton X-100, 150 mM NaCl, 10% glycerol, 5 mM EDTA, 5 µg/ml pepstatin, 1 mM PMSF, 1 mM NaF, 1 mM Na3VO4), incubated at 4 °C for one hr followed by centrifugation at 16,000g for 10 min to collect the supernatant. These samples were directly used for SiMPull. For bulk immunoprecipitation, protein-A beads were added to pre-clean the lysate (2 hr incubation at 4 °C). PKARII antibody was then added to the lysate for overnight immunoprecipitation followed by 1 hr incubation with protein-A beads. Control rabbit IgG was added at final concentration 2 µg/ml. The immunoprecipitated proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes for immunoblot analysis with AKAP150 and PKARII antibodies.
(His)6-tagged PcrA purified protein and cell lysate were prepared as previously described35. The protein was immobilized on slides via antibody against polyhistidine tag. A Cy3 and Cy5 dual-labeled partial duplex DNA (Integrated DNA Technologies, Inc.) with a 5' tail was added to immobilized protein. The sequence of DNA used was: 5'Cy3-(dT)40-GCC TCG CTG CCG TCG CCA-3' + 5'- TGG CGA CGG CAG CGA GGC-3'-Cy5.
We thank Sua Myong, Prakrit Jena, Sinan Arslan and Reza Vafabakhsh for helpful discussions. The expression vector encoding YFP-MAVS gene was a gift from Daniel Lamarre (Université de Montréal). This work was funded by NIH grants (AI083025, GM065367 to T.H.; HL082846 to Y.K.X.; AR048914 to J.C.). Additional support was provided by NSF grants (0646550, 0822613 to T.H.). T.H. is an investigator with the Howard Hughes Medical Institute.
Author contributionsA.J., K.Y.X., and T.H. designed research. A.J., R.L. and Y.I. conducted experiments, R.L., B.R., E.A. and J.P. provided samples, K.R. and Y.I. contributed important ideas to the experiments, A.J. and R.L. analyzed the data and A.J., K.Y.X. and T.H. wrote the paper with inputs from other authors.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.