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Bocote showcases bold patterning and dramatic stripes. Its golden brown backdrop is interspersed with deep chocolate veins and it has a fine texture that polishes beautifully. Despite its distinctive grain pattern, Bocote is delightfully easy to turn. These turning blanks are available in an assortment of sizes for bowls, platters, carvings, bottle stoppers, chess pieces, tool handles, table legs, spindles, finials, candlesticks and whatever else your imagination can conjure up!
Due to high demand and limited supply, we cannot always guarantee that this item will be in stock.
Due to high demand and limited supply, we cannot always guarantee that this item will be in stock.
- Native to Central America and the West Indies.
- Color: Golden brown heartwood with dark stripes; sapwood is pale.
- Grain: Fairly straight.
- Very hard and heavy.
- Extremely resistant to rot and insect damage.
Tech Spec
Customer Reviews
5 Review(s)Average Score:
4.8/5Bocote is a great wood to...
Posted January 2, 2011
Bocote is a great wood to turn, and although it is a hardwood- in my opinion it is a very soft one. I have always been pleased with the Bocote I order from Rockler and have done so numerous times. Frankly, I think their supply of Bocote usually looks much better than other suppliers I have tried. Typically their price for it is also lower.
I don't actually use these blanks...
Posted June 27, 2009
I don't actually use these blanks for turning, I use them to hand carve one off handles for my files and knives, and also for letter openers and key chains for gifts. I also keep pieces on hand for making trim pieces for boxes of all nature. I too find it difficult to drill, but my carving knives and chisels seem to maintain a good edge throughout each piece with only need for stropping between pieces.
This wood turns well but is...
Posted March 7, 2009
This wood turns well but is difficult to drill, you must continually clean the drill bit. It gives off a pleasant aroma when it is worked. It is a very hard wood which works well for my oyster knives. The piece that I recieved has nice pronounced grain. The color is dark brown and one of my knives came out with a very slight green tint, barley noticeable. I would highly recommend this wood.
I was very surprised by Bocote....
Posted October 10, 2008
I was very surprised by Bocote. The grain pattern is awesome. Easy to cut, no nicks just a really good quality wood that's fun to work with.
Wonderful wood to work with, by...
Posted September 30, 2008
Wonderful wood to work with, by far one of my favorites. The grain pattern pops right out with the application of wax. Plus, it smells like dill pickles!


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One of the hottest new ebony pornstars out there today, Pinky XXX (aka Pinky) is turning lots of heads with her pretty face and bountiful ass. Pinky was born June 24, 1980 and got into XXX in 2006 at the age of 26. At just 4'11" with a 34B-26-38 figure, Pinky is mostly booty. A black pornstar, Pinkie gets her catchy name from her pink hair. Pinky is quickly racking up titles, and has even directed two of her own. Profile Link: http://pornstartweet.com/Pinky
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subset: commoncrawl


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WARREN - Bonita Jean Brown, 60, of 2199 Draper St. S.E., died at 3:15 p.m. Sunday, Dec. 2, 2012, at Northside Medical Center following complications of heart disease.
She was born March 6, 1952, in Warren, the daughter of Betty Williams, and was raised by her grandparents, Eugene and Odell Johnson Broadus.
Mrs. Brown was a clerical worker with the Veterans Administration Hospital and was a member of Parkman Road Church of Christ.
Article Photos
Survivors include two sons, Brian and Michael, both of Winona, Minn.; one brother, Audie Broadus of Warren; two sisters, Cordella Thomas of Columbus and Jennifer Thomas of Warren; and three grandchildren.
She was preceded in death by her husband, Allen O'Neal Brown.
Calling hours will be from 11 a.m. to noon Tuesday at Elim Christian Center, where services will be held at noon. The family will receive friends at 2199 Draper St. S.E., the family home.
Arrangements were handled by the Sterling-McCullough Williams Funeral Home.


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Going outside to see what kind of snow I have to deal with , and found out that my drive is drifted shut! Live out by HWY30 and the apartment complex faces North/South!
Winter Event 01/12/12
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Agenda, Handouts, and Other Documents
Session Handouts for Days 1 and 2 (Mon & Tue, June 25-26)
Session Handouts for Days 3 and 4 (Wed & Thu, June 27-28)
These calls are provided by the WDE Special Programs Division each month. The purpose of the calls is to provide guidance to professionals working with children and youth with disabilities. The information is intended to assist districts and public agencies to achieve compliance with the Individuals with Disabilities Education Act, Wyoming Education Rules Chapter 7 and Special Education policies and procedures. Calls are scheduled on Thursdays from 3:00 p.m. to 4:00 p.m. on the dates listed below. The call in number is (877) 278-8686 and the participant PIN is 095102.
2011-2012 Technical Assistance Calls
2010-2011 Technical Assistance Calls


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PROXY: 'Who Are You?' EP Out Now on Dim Mak Records
The audio onslaught continues with the WHO ARE YOU? EP, the latest release from world-renowned Russian producer PROXY out now on Dim Mak Records in North America and on Turbo Recordings internationally. The WHO ARE YOU? EP continues the saga of PROXY's MUSIC FROM THE EASTBLOCK JUNGLES series, his two-part debut album out now, and features the title track along with remixes from buzzing producers/DJs Pilo, Attaque, Trumpdisco and The Sneekers.
The EP kicks off with PROXY's "Who Are You (770EQ)" from MUSIC FROM THE EASTBLOCK JUNGLES PART II. Earlier this month, PROXY teamed up with MTV Hive to premiere the WHO ARE YOU? EP in full. SaysSteven Aguiar of the track, "Here, Proxy's up-tempo title track is carried by an alternating kick-snare and an adrenalizing low-end chomp, the female sample in between drops reinforcing the song's bad-ass attitude."
Los Angeles' PILO continues the chaos with his remix, bringing a deep bass barrage of hard house, whileEssex-based producer Attaque slices through a vast concrete jungle with his reworked version. Australian duo Trumpdisco goes full throttle on their electro-smashing revision and Lithuanian electronic music duo The Sneekers closes out the EP with their space odyssey-inspired take on the track.
Earlier this year, PROXY released the MUSIC FROM THE EASTBLOCK JUNGLES series in two parts to critical acclaim. The album, a dark reflection of PROXY's grim and oppressive world, harnesses the spirit of pure electronic music culture and showcases the prolific underground dance floor-czar's wide-ranging body of work. The MUSIC FROM THE EASTBLOCK JUNGLES series is the product of an artist who forged a sound in complete isolation with visa problems leading him to entrench himself in the middle of nowhere in his nativeRussia, continuing in the U.K. rave tradition of his Essex-spawned idols, The Prodigy. The disparate pieces ofMUSIC FROM THE EASTBLOCK JUNGLES converge into a monolithic nation-statement, one worthy of one of the most talented producers of his generation.
The complete track listing for the WHO ARE YOU? EP is as follows:
- Who Are You? (770 EQ)
- Who Are You? (Pilo Remix)
- Who Are You? (Attaque Remix)
- Who Are You? (Trumpdisco Remix)
- Who Are You? (The Sneekers Remix)
The WHO ARE YOU? EP is available now at iTunes (http://itun.es/i6JM9Fp) and Beatport (http://btprt.dj/ZxFHs8).


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A collection of news and information related to High School Musical 3 (movie) published by this site and its partners.
Top High School Musical 3 (movie) Articles
Displaying items 34-44
'Hannah Montana' Movie Moves to April
Story | Sep 23, 2008Fans will get a glimpse of Hannah Montana's first trip to the big screen a little bit earlier than expected. Disney announced on Monday (Sept. 22) that the fittingly titled "Hannah Montana: The Movie" will now open on April 10, 2009, three weeks... -
'High School Musical's' Vanessa Hudgens buys Studio City home for $2.75 million
Movie review: High School Musical 3: Senior Year -- 3 out of 5 stars
'High School Musical 3: Senior Year'
Story | Oct 24, 2008Call it a musical with training wheels, call it corny, call it Old-Older-Oldest School -- as in Judy Garland and Mickey Rooney and "Let's put on a show!" But "High School Musical" makes the journey from The Disney Channel to the big screen with its "Gee... -
Zac Efron's 'Footloose' Dancing Forward
Story | Oct 28, 2008Hot on the heels of the $42 million opening for " High School Musical 3," a Zac Efron-centered remake of "Footloose" is now on the fast-track. The project was first announced in July of 2007, but fell behind "17 Again" and "HSM3" on Efron's slate while... -
International Audiences Bond with 'Quantum of Solace'
Story | Nov 3, 2008Although it won't open in the United States until Nov. 14, "Quantum of Solace" is already breaking international records. Daniel Craig's second tour of James Bond duty made an estimated $38.6 million overseas in the weekend ending Sunday, Nov. 3, despite... -
New James Bond Pic Finds 'Solace' Atop Foreign Box Office
Story | Nov 10, 2008Still awaiting domestic release, "Quantum of Solace" is already a worldwide sensation. Playing in 60 countries on 9,870 screens, the latest James Bond thriller pulled in a whopping $106.5 million at the foreign box office for the weekend ending Sunday,... -
Cheetah Girl to Sue Over Stolen Semi-Nude Pictures
Story | Nov 10, 2008Another Disney Channel star has had illicit photos hit the Internet. Won't they ever learn? Adrienne Bailon, one of the Cheetah Girls, plans to take legal action after racy pictures of her stolen off her laptop have been leaked online, reports TMZ.com.... -
'High School Musical 4' Script in Development
Story | Apr 9, 2008More Wildcats are on the way. It's just a question of which ones. Disney Channel announced that it already has "High School Musical 4" in the works, even though the big-screen "High School Musical 3: Senior Year" won't hit theaters until late October.... -
Ashley Tisdale Remakes 'Teen Witch,' Revisits Mall Past
Story | Apr 17, 2008Ashley Tisdale has big post-"High School Musical" graduation plans, and it doesn't involve college. The 22-year-old will star in the revamp of United Artists' "Teen Witch" as well as executive produce a project about the mall for the Disney Channel,... -
Lachey to host 'High School Musical' reality show


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Photo by Hank Lohmeyer Traffic, calm but congested, lines up in both directions, blocking the southbound lane of Highway 65 at Cedaredge Elementary School one day last week. Traffic trying to access the student drop-off/pick-up point is the reason for the congestion.Cedaredge Police Chief Robert Yant reported to the town board during its Feb. 21 meeting on a series of safety and security response meetings that he and his officers have had recently with local school officials.
The meetings have been geared to establishing procedures and for coordination of handling emergency situations that could arise, including even the presence of "active shooters" on school grounds, the chief explained.
Read more...
Surface Creek
Photo by Hank Lohmeyer Dozens of middle school students from Hotchkiss and Delta got a day of outdoor fun learning on Grand Mesa last week as they participated in the U.S. Forest Service Junior Snow Ranger Program. Above, Anita Evans of Hotchkiss Middle School instructs some students on assembly and setup. Transportation for the students and teachers was paid for by Gunnison Energy Corporation.It was a big week and a big weekend for educational and recreational activities on Grand Mesa.
From midweek through the weekend, individuals, groups, schools, government, outdoor recreationalists and a local chamber of commerce chose mesa top venues for their various activities.
Page 237 of 427
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Private Dining
Farmstead at Long Meadow Ranch
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Reviews ›
738 Main Street
Saint Helena, CA 94574
St. Helena
Getting there ›
$30 and under
More Details ›
Farmstead at Long Meadow Ranch Details
Dining Style:
Casual Dining
(707) 963-9181
Hours of Operation:
Monday-Thursday 11:30am-9:30pm
Friday-Saturday 11:30am-10:00pm
Sundays 11:00am-9:30pm
Payment Options:
AMEX, Discover, MasterCard, Visa
Executive Chef:
Stephen Barber
Dress Code:
Casual Dress
Accepts Walk-Ins:
Additional Details:
Banquet/Private Rooms, Bar Dining, Bar/Lounge, Beer, Farm to Table, Fireplace, Full Bar, Gluten-free Menu, Happy Hour, Non-Smoking Restaurant, Patio/Outdoor Dining, Personal wines welcome, Private Room, Takeout, View, Weekend Brunch, Wheelchair Access, Wine
Off-site catering available. Please contact the Events department at email@example.com or (707) 963-4555 ext 7210.
Live Music every Friday at the Outdoor Bar starting at 5:00pm
Special Events & Promotions:
Weekly Traditions include:
Monday Family Night : Meatloaf - Comfort food and kid's specials
Tuesday Fried Chicken Night - Crispy fried chicken
Wednesday Nights : California Creole - Creole favorites
Friday Live Music - Starts at 5:00pm at the Outdoor Bar
Sunday Brunch - Rustic brunch starts at 11:00am
Getting there
Cross Street:
Charter Oak
Private Lot
A word from Farmstead at Long Meadow Ranch
Farmstead at Long Meadow Ranch showcases the seasonal, ingredient driven American farmhouse cooking of Stephen Barber. The ingredients in the Farmstead kitchen are foraged from local sources and feature Long Meadow Ranch's all natural grass-fed beef, organic and sustainably produced eggs, fruits,
vegetables, extra virgin olive oil, and honey - as well as ingredients from local artisan producers.
Foodspotting photos
See all photos >
Awards voted by OpenTable diners
Diners' Choice Winner
Diners' Choice Winner
Good for Groups
Diners' Choice Winner
Great for Lunch
Diner Reviews
This was my first visit -- I will
return! I started with the lacinato kale salad with chile pequin, lemon dressing and toasted parmesan -- truly outstanding! My main was the California arborio rice with sauteed mushrooms, winter squash, broccolini and parmesan, also outstanding! I enjoyed a glass of Farmstead's own pinot noir...perfect. The server was especially wonderful and patient with my diva dining companion....and for that I was most grateful. After lunch, I walked through the gardens where the chefs were picking fresh everything for the dinner menu. Also enjoyed seeing the beautiful espalier apple trees in bloom."
Dined on 3/4/2014
Super fresh farm to table
restaurant, served an amazing beet salad and cheeseburger. A really great stop in St. Helena."
Dined on 3/8/2014
Convenient Central Valley location
in St. Helena. Corkage is only five bucks and it goes to charity. We just had sandwiches, which were super, but not enough of a test to award a higher rating."
Dined on 3/7/2014
Really great food, once again, and
served by an attentive, happy staff. Would recommend to anyone as best place in Napa Valley for a great, local dining experience...at a good price. Great drinks and wines as well."
Dined on 3/4/2014
We went there for their Mardi Gras
Fat Tuesday but go there a lot. Like to go for lunch with girlfriends. So the only negative is the prices on the wines. way over priced. if you look around- very fw diners are drinking a bottle of wine. Just wine by the glass and most are drinking the house Long Meadow Ranch who owns Farmstead and their wines are marginal against such a great menu. So hint- corkage is only $5.00 and it is donated to a local charity."
Dined on 3/4/2014
Had to wait almost a half hour
even though we had a reservation. The food was more like what one would expect at a fast-food joint -- definitely not worth the money or wait."
Dined on 3/4/2014
The food is terrific and the
service is outstanding!"
Dined on 3/3/2014
I frequent this restaurant and
have always enjoyed it.. I will say I miss the butterscotch bread pudding but can't complain a great place to dine."
Dined on 3/1/2014
We enjoyed our experience very
much! Love the farm-to-table concept. Outstanding service and food alike. Thank you for making our experience special, especially with my picky vegan sister."
Dined on 3/2/2014
Great place for a couple's lunch.
Try the Gnocchi!!"
Dined on 3/1/2014
We thoroughly enjoyed our dinner
at Farmstead. One of the best burgers ever, great steak tartare, and charcuterie, and the best St. Louis ribs since I lived in St. Louis many years ago! The new DIY smores were a real hit, though we wondered if the restaurant was a little crazy to give people hot coals... All in all, a very enjoyable evening."
Dined on 3/1/2014
In Napa Valley. They grow their
own vegetables. Every dish we had was great. We ate their twice and we're served by Molly who was as professional as they come"
Dined on 2/28/2014
We ordered the pork chop with
grits, cheesy bread, meatballs, or burger. They were all delicious. The pork chop was cooked to perfection; juicy and tender on the inside, and crispy around the edges!"
Dined on 2/27/2014
Really enjoyed our dining
experience. Would Def return and recommend to friends"
Dined on 2/27/2014
Fun, good food, good variety.
Nothing fancy, but very good"
Dined on 10/13/2013
Food was good but not outstanding.
We were expecting much more. Noise level was unbearable."
Dined on 11/2/2013
Nothing special. Food was mediocre
service was not outstanding and price was higher than expected for meals purchased"
Dined on 2/25/2014
I am a pretty picky eater and love
a place that has fresh veggies with a ton of flavor...THIS IS IT! The kale salad was beyond amazing and our waitress was a doll! She gave me the dressing recipe! I also loved the trout. My daughter had the sole and wasn't a fan but everything else was 5 star!"
Dined on 2/24/2014
Great food, service, energy. We go
to Farmstead for their use of local, seasonal food and they never disappoint. I've never had the burger before, and now it will be a staple. Cooked to perfection with oodles of condiments, including arugula on a pillow-soft potato bun. Kale salad was tasty with Parmesan crisps, altho I wish it was chopped a tad finer. Wines are carefully selected and unique. And the nominal corkage goes towards charity."
Dined on 2/24/2014
Great place off 29. Rustic
atmosphere, fresh food, interesting menu."
Dined on 2/21/2014
My friends and I went there for a
Sunday Brunch. The food was fresh and delicious. I also like that we did not have to pay a "fixed" price for the brunch. So we did not over eat or end up with too much food. I definitely recommend Farmstead for special occasions like Mother's Day or Birthdays. I will definitely go back to this restaurant."
Dined on 2/23/2014
Burger with fried egg, grilled
artichoke and a pilsner, a little bit o heaven! We look forward to our annual farmhouse fix. It was well delivered."
Dined on 2/23/2014
Burger was disappointing. No
condiments provided for a little crunch. Overcooked. Lots of breakfast - style potatoes so felt like a giant carbo load rather than a more balanced plate. Pork sandwich was ordinary but tasty. Ribs appeared a bit tough. Was hoping for more given the hype."
Dined on 2/21/2014
This is the quintessential comfort
food venue. Great American food and great service. A gem in the Napa Valley."
Dined on 2/20/2014
This was recommended by a few
friends who had been to Napa and they were right. The food was outstanding, the service was great, and the ambiance was special. Well worth the visit!"
Dined on 2/19/2014
The food was so good and fresh.
Prices were reasonable and the set-up (old barn) was very cool. Nice restaurant. Would return."
Dined on 2/18/2014
Such a great experience. The food
was incredible. Their homemade hot sauce was fantastic. I would recommend this place to anyone."
Dined on 2/18/2014
We went here after a hike to
devil's punch bowl. We couldn't be more pleased with the food and the general experience."
Dined on 2/17/2014
This was probably our 5th time at
Farmstead, so I'm going to be a bit picky. 1st - looks like reservations are booked too closely, 5 parties waiting to be seated at the same time, we waited 15 min. Some kind of bread product should be offered and not charged for. All of our food came out just warm, using plates that aren't being warmed so the food cools down too quickly. Food is tasty and well prepared. Staff was attentive and on top of the service. $5.00 corkage and that's being donated to a local non-profit. Very nice wine list. I'm a local and go out to dinner 2 times a week. I'm not in a rush to return, but I will."
Dined on 2/16/2014
Farmstead and the whole Long
Meadow Ranch experience was incredible. From the estate tour, to the wine and olive oil tastings, to the restaurant itself, we couldn't have had a better time. All the staff were so friendly, the food was delicious, and we enjoyed every second of it. Thanks!"
Dined on 2/14/2014
Wasn't disappointed at all! It was
actually beyond my expectation!"
Dined on 2/15/2014
The food is incredible and the
atmosphere is perfect. I highly recommend Farmstead."
Dined on 2/16/2014
Had a great time - we got a last
minute reservation here and couldn't have been happier. The staff was so friendly and very helpful with guidance on the menu. I wouldn't have normally ordered a vegetable appetizer, but since everything is freshly grown on site and came highly recommended, we were eager to try it and it was delicious. My filet could not have tasted any better - top 5 filets I've ever had. The cheddar biscuits were also some of the best I've ever had - the honey butter was delectable. And, last but not least, the pie was amazing. The graham cracker crust and chocolate cream were again - to die for. Loved it all!"
Dined on 2/15/2014
Good was definitely excellent,
servers were friendly. Layers of flavors, food wasn't boring at all."
Dined on 2/15/2014
I am not sure how they do it, but
they blend home style rustic cooking with a hip vibe. Food is amazing, portions are generous and the feel is "welcome home". I never visit wine country without a trip to Farmstead."
Dined on 2/15/2014
Too loud. Food was just okay. Not
a relaxing meal."
Dined on 2/15/2014
From vegetarian to major meat
eaters, this is a fabulous place. Smoked meats exceptional. Great for a group with different diets. Wonderful farm-to-table and sustainable farming practices. Good wine. Service very friendly and helpful."
Dined on 2/11/2014
Met a client to discuss a local
project and it was perfect for the meeting. The food was great, the noise level was active but not overbearing and the service was outstanding. Emily was very attentive, but knew when not to interrupt us. Will definitely go back for both personal and business. Thank you"
Dined on 2/6/2014
What a lively, unique environment
this restaurant provides. Lots of energy, delicious menu full of hometown creative options served. We had a blast!"
Dined on 2/8/2014
This restaurant has fabulous
eclectic food and beverages. So good we went Saturday for dinner and Sunday for brunch. Risotto with asparagus and mushrooms, grilled artichoke for appetizer was to die for!!! The hash on Sunday had beets which truly sparked the meal. I highly recommend this restaurant and really you must have a reservation. You can take a chance with the bar, but it's a small number of seats. They actually make their own ketchup and Tabasco and it's fabulous! Recommend the socialite drink. Has a nice spice to it. Bloody Mary's could use a bit more kick, so if that's your thing, ask for them spicy."
Dined on 2/8/2014
More reviews ›
Easter Brunch & Dinner Reservations
Sunday, April 20, 2014
Serving Brunch, Lunch and Dinner. Enjoy the Farmstead benedict, beef hash, biscuits&gravy, cheeseburger, carefully selected vegetable board and mimosas or bacon bloody marys.
Send a Gift Card for Farmstead at Long Meadow Ranch
Buy now and deliver it instantly by email, or print a beautiful card to give in person.
Buy one today! ›
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Diners' Choice Awards
Wine Country Winners
Good for Groups
Great for Lunch
Outdoor Dining
Most Booked
Learn more ›
Like Farmstead at Long Meadow Ranch
Plan your next party here
We offer two private dining venues with a range of six different options for any size group. Located in beautiful and
historic St. Helena, we are perfectly positioned in the heart of Napa Valley.
Great for events up to:
More details and contact info ›
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My contacts are not appearing in BBM
If you saved a backup file of your contact list to your smartphone, on a media card, or to your computer using the BlackBerry® Desktop Software, you can restore your contact list using that backup file. Depending on your organization, if you previously associated an email address with a smartphone on which the latest version of BBM™ was installed, you might be able to restore your contact list over the wireless network. Research In Motion recommends that you regularly create and save a backup file on your computer, especially before you update any software. Maintaining a current backup file on your computer might allow you to recover smartphone data if your smartphone is lost, stolen, or corrupted by an unforeseen issue.


text:
911 Incidents
This page is designed as a hazard and traffic avoidance warning system, and is NOT meant as an avenue to obtain police reports or to use for statistical analysis. Contents of the webpage are not archived. Please do not call 911 or the ECD offices with inquires regarding current or past entries on this website.
Last Updated 3/12/14 12:29am
Note: 9-1-1 incidents can only be plotted on the map if they are reported by cellular phone.
Subscribe to the 9-1-1 Incidents RSS Feed
Incident | Status | No. | Time |
Hit and Run, no injury and no blocking 2345 MARKETPLACE DR, Henrietta |
ONSCENE | MCOP140710054 | 03/12/14 12:22am |
Hit and Run, no injury and no blocking
39 N CHURCH RD, Greece show on map |
ONSCENE | GREP140710019 | 03/12/14 12:06am |
Any dumpster, grass or rubbish fire not posing an exposure problem 185 EXCHANGE BL, Rochester |
ONSCENE | CTYF140703561 | 03/11/14 10:31pm |
Human life endangered by animal
64 THOMAS ST, Rochester show on map |
DISPATCHED | CTYP140703521 | 03/11/14 10:10pm |
Hit and Run, no injury and no blocking BROWN ST/KENSINGTON ST, Rochester |
ONSCENE | CTYP140703389 | 03/11/14 9:14pm |
Incident Map
911 Incident
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Youngpoong Bookstore (영풍문고) | ||||||||
| ||||||||
Introduction | ||||||||
Youngpoong Bookstore has six branches as well as an on-line bookstore. Of those, the most famous branches are the Jongno and the Gangnam stores. Since both places are connected to the subway station, the location is very convenient. The Youngpoong Bookstores are not ordinary bookstores. They are exclusive cultural centers that carry all kinds of cultural products. The Jongno branch, located in the Youngpoong Building is spread out over two floors. There are roughly one million books to choose from here. Various kinds of stationary are also available and you can also enjoy fast food at several adjoining restaurants. At the record shop you can listen to the latest Korean hit songs. Since you can reserve tickets there for events and performances, if you see something on the posters that interest you, you can purchase them on the spot. At the large bookstore you will find quite a number of people reading books. Facilities for visitors such as photocopy machines, a gift-wrap center and photography studios are on hand. Outside the bookstore, the streets are directly connected to the areas of Jongno and Euljiro. As one of the most intricate places in Seoul, Jongno offers many places to eat and enjoy oneself. Euljiro has so many buildings that it actually seems like a forest of buildings. If you follow these buildings you will soon find yourself in the area of Myeongdong, another Mecca for shoppers. The Youngpoong Book store in Gangnam is located in the basement of Central City's Young Plaza, the area's giant shopping mall. It is 3600 pyong (pyong = 2.2 m2), roughly 600 pyong larger than the Jongno store. It provides all the latest cultural information and features a book café, event hall, magazine plaza, and information center. Even those not wishing to purchase any books can still spend time enjoying the atmosphere. Central City Plaza also has a lot to offer visitors seeking enjoyment: a department store, movie theater, hotel, car exhibition, game room, record shop, sauna, international conference rooms, bank, post office, medical center, and food courts. Therefore if you feel bored staying inside the bookstore, you can always get out and enjoy the many other activities on hand. The Express Bus Terminal is also connected to the bookstore. You may want to buy a book for your bus ride to the countryside. |
Subsidiary Facilities | ||||||||
Jongno - parking lot, restrooms Gangnam - parking lot, restrooms, benches |
Admission / Participation Fees | ||||||||
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Hey, just wondering if you plan on keeping your Amoonguss/Morobareru analysis. If not, can I take it over? Thanks, ~Moo
Yeah i suppose..But the thing is its not very hard to take down Ranky when its weakened because its slow as fuck and almost all offensive teams pack decently hard hitters to kill it at a weakened state (Such as Skymin/Rotom/Tar for Iconics team).Its more of an annoyance because youl almost never Ohk0 it and it will generally kill something off when you try to whittle down its Hp..But yeah still works i guess =p
Regarding your reply in Iconics Team...Terakion can only do about 35-40%'ish dammage to CM Rankurusu so its not exactly even a decent check to that thing.
I just felt a need to comment on the intro to your RMT, lol that is definitely one of my favourite songs and I'm glad that my good taste is rubbing off on SOMEBODY. I can't tell if you know that because of my recent RMT or if you simply inferred it by chance from my name, which would actually be pretty cool and a rather funny coincidence. I almost made my name Morrisey when I changed my name from Toxic Spork, the lead singer of the Smiths.
the apprentis progrom hurts my head ... i would want to sighn up to see if im any good right , but on the other hand its like bleh
lol I've never been there... they must think some pretty insane crap like hyper beam is DA BOMB on gyarados I'm kinda sad now lol they may delete it :/ I worked hard on that thing
Nice RMT you had there! I had one nitpick though, since you maxed out your Sheddy's Defense and HP I would make it a more stall oriented set, something a little like. Shedinja @ Flame Orb Impish Nature 252 HP, 252 Def, 4 Sp. Def Wonder Guard - Only super effective attacks harm this Pokémon ~Dig ~Protect ~Substitute ~Rest / Toxic I'd recommend black sludge but that would violate the item clause, but here is the standard staller set. Sub up, and use dig and protect to stall, then rest to recover damage. The flame orb is a nasty trick to offensive pokemon (especially physical ones) looking to limit your walling with Trick.


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will normally automatically cross file system boundaries in
order to archive files which are part of a directory tree. You can
change this behavior by running tar
and specifying
'--one-file-system'. This option only affects files that are
archived because they are in a directory that is being archived;
will still archive files explicitly named on the command line
or through '--files-from', regardless of where they reside.
Prevents tar
from crossing file system boundaries when
archiving. Use in conjunction with any write operation.
The '--one-file-system' option causes tar
to modify its
normal behavior in archiving the contents of directories. If a file in
a directory is not on the same file system as the directory itself, then
will not archive that file. If the file is a directory
itself, tar
will not archive anything beneath it; in other words,
will not cross mount points.
This option is useful for making full or incremental archival backups of a file system. If this option is used in conjunction with '--verbose' ('-v'), files that are excluded are mentioned by name on the standard error.
6.10.1 Changing the Working Directory | Changing Directory | |
6.10.2 Absolute File Names |
This document was generated by Sergey Poznyakoff on October, 20 2013 using texi2html 1.78.


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Photographer Robert Emrick Collection Northampton photographs Date of photo March 19, 1936 Description High Water. View of Main Street from Bridge St. Film: Ver. 620 Kodak Object ID EM 620-335 Object Name Negative, Film Place Main St. and Bridge St., Northampton Title High Water
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Benny Lawrence Zane
A memorial service for Benny Lawrence Zane will be held at 2 p.m. Saturday, Aug. 7, at the Church of Jesus Christ of Latter-day Saints, 815 W. Washington St., Sequim. Bishop Holloway of the Happy Valley Ward will officiate.
Mr. Zane died July 24, 2010, at the age of 79.
He was born April 28, 1931, in Protection, Kan., to Ralph Crittenden and Florence Sara Lawrence Zane.
He enlisted in the United States Navy shortly after graduating from Protection High School in 1949.
He earned a Bachelor of Science degree from the University of Kansas in 1956 and a master's in business administration from the University of Illinois - Champaign-Urbana in 1957. He worked toward a Ph.D. at the University of Southern California.
Mr. Zane held marketing positions with I.B.M., Caelus Data Products and Telex and served as director of financial management for the federal government.
One of his great loves was football, and he had experience as a football and wrestling coach at both the high school and university levels.
He is survived by his wife of 10 years, Carol Ann Cook Zane; sons F. Kelly Zane of Flower Mound, Texas; R. Scott Zane of Hutchinson, Kan.; and J. Anthony Zane, also of Kansas; daughters and son-in-law Kellan and David Bruce of Poulsbo, and Kaleigh Zane of Seattle; and four grandchildren.
He was preceded in death by his brother Robert.
Memorial contributions may be sent to the Volunteer Hospice of Clallam County, 540 E. Eighth St., Port Angeles, WA 98362.
Alice B. Lauderback
A memorial service was held for Sequim resident Alice B. Lauderback, who died July 22, 2010, at the age of 78 of pancreatic cancer.
She was born in Sandpoint, Idaho, to Alexander and Zelma Lula O'Neal Bocook on Jan. 28, 1932.
One in a family of 18 children, she was baptized in the General Assembly and Church of the First Born in 1951.
She married Bert Lauderback in 1950. They had five children. The marriage ended in divorce in 1971. She moved to Sequim from Spokane in 1974 and worked as a cook until opening Alice's Wonderland Daycare in 1986, where she stayed until her retirement in 2001.
After retiring, she moved into Suncrest Apartments, where she enjoyed organizing events. She also enjoyed doing crafts, sewing and singing.
She is survived by her sons and daughters-in-law Ken and Cheryl Lauderback and Les and Cindy Lauderback; daughters and sons-in-law Ina and Dale Turton, Kathy and Theodore McDonald, and Gayla Johnson; siblings David Bocook, Enice Bocook, Kenneth Bocook and Rose Sevier, all of Spokane; Helen Holbrook of Olympia; Gladys Minty of Sequim; and Ina Sams and Nancy Sams of Spokane; 15 grandchildren; 13 great-grandchildren; and one great-great-grandchild.
She was preceded in death by brothers Harvey, Jeff, Johny, Harry, Willie and Tom; sisters Annie, Mary, and Lucy; and great-grandson Jacob Graves.
Memorial contributions may be sent to Volunteer Hospice of Clallam County, 540 E. Eighth St., Port Angeles, WA 98362.
Leslie Alice Peeples Ovesen
A memorial service will be held at a later date for Sequim resident Leslie Alice Peeples Ovesen, who died July 19, 2010, at the age of 75.
She was born June 8, 1935, in Missouri, to Leslie and Ruby Taylor Peeples. Raised in Southern California, she also lived in Sacramento, Calif., Lake Tahoe, Calif., and Las Vegas, Nev. She married Carl Erick Ovesen on Apr. 26, 1995.
She is survived by her daughter Laurie Willey of Warrenton, Ore.; brother Roger Peeples of Woodland, Calif.; a granddaughter, three great-granddaughters and two great-grandsons.
She was preceded in death by her son Michael Young and daughter Patricia Young.
Norman Miner
A memorial service will be held at 1 p.m. Saturday, Aug. 7, at Sequim Community Church, 950 N. Fifth Ave., Sequim, for Sequim resident Norman Miner.
Dr. Scott Koenigsaecker is the officiant.
Mr. Miner died July 17, 2010, at the age of 91.
He was born Nov. 15, 1918, in Wakefield, Neb., to Wiliam and Joreta Turner.
He worked for the Army in the aircraft industry in California during World War II.
He married Lois Miner on Nov. 6, 1966, in Garden Grove, Calif. Until his retirement at the age of 55, Mr. Miner was the manager of the Orange Drive-In Theater in Orange, Calif. After retirement, the Miners moved to Happy Valley in Sequim, where he built their home. He enjoyed gardening, raising cattle, running a wood-kindling business, and fishing.
He is survived by his wife, Lois Miner; sons Bill Miner and Tom Miner, and daughter Sue Brown, all of California; stepchildren Kathleen Perez of Loomis, Calif., and John Bachelor of Reno, Nev.; five grandchildren; three step-grandchildren; and three great-grandchildren.
Charlotte Hall Cook
Charlotte Hall Cook died July 30, 2010, at the age of 94.
She was born April 26, 1916, in Buhl, Idaho, to Cassius and Sarah Morris Woodward.
No additional information was available.
Sequim Valley Chapel was in charge of arrangements.
March 15, 1920 ~ July 14, 2010
Sequim resident Clifford Carlson passed away peacefully on July 14, 2010. He longed to be with his beloved partner in life, Margit... who passed away just 8 months earlier. "Sea Fever" by John Masefield was Clifford's most loved poem and by this you will know him:
I must go down to the seas again, to the lonely sea and the sky, and all I ask is a tall ship and a star to steer her by; and the wheel's kick and the wind's song and the white sail's shaking, and a grey mist on the sea's face, and a grey dawn breaking.
I must go down to the seas again, for the call of the running tide is a wild call and a clear call that may not be denied; and all I ask is a windy day with the white clouds flying, and the flung spray and blown spume, and the seagulls crying.
I must go down to the seas again, to the vagrant gypsy life, to the gull's and the whale's way where the winds like a whetted knife; and all I ask is a merry yarn from a laughing fellow-rover, and quiet sleep and a sweet dream when the long trick's over.
Memorial contributions may be made to: Friends of Liberia.
September 10, 1919~July 9, 2010
Delores Boyd, 90, died peacefully surrounded by her family on July 9, 2010 at Martha & Mary Health Services in Poulsbo.
Delores was born on September 10, 1919 in Irma, Wisconsin to Albert & Margaret Batchelder, the 7th of 13 children. Delores was married to Everett Robarge for 8 years. In August of 1948, Delores married Hollis Avery in Sultan, WA. In 1950 they moved to Sequim and in 1963 moved their family to Port Angeles. Hollis passed away in 1991 after battling cancer. Delores married Art Boyd, a long-time family friend, in April 22, 1995. They enjoyed seven wonderful years together having many adventures. She was diagnosed with a form of dementia in 2002. Delores was moved into Dungeness Courte Memory Care in 2005. After 2 years she moved to Martha & Mary Health Services where she was taken care of with great love and compassion by many exceptional nurses and caregivers.
Delores was a member of the First Presbyterian Church, the Senior Citizen Center, and a lifetime member of the Mountain View Homemaker Club. She was an avid doll collector and a founding member of Potpourri Doll Club. She worked at Shirley's Doll House, volunteered at the Clallam County Museum, and worked on the election board. Many children and adults enjoyed her wonderful storytelling at the North Olympic Library during her years as the Children's Librarian.
She is survived by her spouse, Art Boyd; her children - Faith Robarge Ohlson (Curt), Rita Frick, (Steve) and Ted Avery (David Jones); three grandchildren, seven great-grandchildren and her sister Ila Mae Buchanan. Step children; Tom Boyd, Sandra Rouse, Carl Boyd, and Janis Cobb and their families. Numerous nieces and nephews and was considered "Mom" by many.
A memorial service will be held at the First Presbyterian Church on August 7, 2010 at 1 P.M. Harper-Ridgeview Funeral Chapel, Port Angeles made the arrangements. Please visit the online guest book at www.harper-ridgeviewfuneralchapel.com.
In lieu of flowers, memorial contributions may be sent to
The North Olympic Library System; 2210 South Peabody St.,
Port Angeles, WA 98362 or the First Presbyterian Church;
139 West 8th St., Port Angeles, WA 98362.
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U.S. Soccer Coaching Symposium to Be Held June 3-4 in Conjunction with USA's Match against Spain in Foxborough, Mass.
U.S. Soccer's Coaching Education Department will host a Coaching Symposium on June 3-4 in conjunction with the U.S. Men's National Team's match against 2010 FIFA World Cup Champion Spain in Foxborough, Mass.
April 19, 2011
© U.S. Soccer
"A" Licensed Coaches Earn Two CEUs for Participating;
Coaches will Attend a U.S. MNT Training Session and the Match on June 4 against Spain
CHICAGO (April 19, 2011) – U.S. Soccer's Coaching Education Department will host a Coaching Symposium on June 3-4 in conjunction with the U.S. Men's National Team's match against 2010 FIFA World Cup Champion Spain in Foxborough, Mass.
The U.S. Men's National Team will face Spain on June 4 at Gillette Stadium in Foxborough, Mass. Kickoff against the No. 1-ranked team in the world and defending European Champion is set for 4:30 p.m. ET, and the match will be broadcast live on ESPN and Univision. Fans can also follow the match live on ussoccer.com's MatchTracker and on Twitter @ussoccer.
Registration for the symposium includes two days of educational presentations and discussions, an opportunity to observe U.S. MNT training session, a match ticket, a pre-game reception and a U.S. Soccer CoachesNet scouting and training notebook. Cost for the symposium is $240 for active U.S. Soccer CoachesNet members and $250 for non-members. Registration is open to all coaches.
The high-profile friendly takes place three days before the U.S. begins group play in the 2011 CONCACAF Gold Cup, where they join Group C of the 2011 CONCACAF Gold Cup along with Canada, Panama and Guadeloupe.
"This is an exciting time for U.S. Soccer as the No. 1 team in the world visits the Boston area," said U.S. Soccer Director of Coaching Education Dave Chesler, who will oversee the symposium. "This symposium will give coaches a unique opportunity to examine the USA's final preparations prior to a major international competition."
- Join CoachesNet Now! | Download Symposium Application SOLD OUT
U.S. Soccer "A" licensed coaches will earn two "Continuing Education Units" for attending this symposium and completing a written technical component related to this match.
U.S. MNT vs. Spain Coaching Symposium Tentative Schedule
Date Time (ET) Event
June 3 2-2:45 p.m. Registration
June 3 3-4:45 p.m. Presentation
June 3 5 p.m. Observe Training
June 3 7 p.m. Presentation
June 4 12-2:30 p.m. Presentations
June 4 2:30 p.m. Pre-game Reception
June 4 3:30 p.m. Observe on-field warm-ups
June 4 4:30 p.m. U.S. Men's National Team vs. Spain
June 4 7 p.m. Post-game Match Analysis
Schedule is subject to change. Times may be adjusted to accommodate changes in training schedule and coaching staff. More information will be available in due course.
Questions regarding the symposium can be directed to Bryan Koch in the U.S. Soccer Coaching Department at email@example.com or 312-528-1219.


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Two robbers riding black BMX bikes set upon a victim in Yarmouth.
Monday, September 17, 2012
A robbery victim was set upon by two BMX biking robbers and grabbed by the throat while cash was stolen from his bag.
In what police have described as "a nasty robbery which left the victim shaken by the aggressive nature", a 17-year-old boy was set upon by two male youths on black BMX bikes just as he turned into Churchill Road from Estcourt Road, by Northgate Hospital, Great Yarmouth, between 10.15pm and 10.30pm on Friday, September 14.
He was grabbed by the throat by one male whilst the other stole £50 cash from his bag. The robbers were then seen to cycle off along Estcourt Road in the direction of Northgate Street.
The first offender is described as a black male, 17 to 18-years-old, five foot eight inches tall, and was wearing a green woolly Adidas jacket and riding a black BMX Bike.
The second is described as a black male, 17 to 18-years-old, five foot 11 inches tall, and was wearing a red and white Adidas jacket and was also riding a black BMX Bike.
DC Kevin Maskell from Great Yarmouth CID said: "Although the victim was uninjured, this was a nasty robbery which left him shaken by the aggressive nature. We are treating it as an isolated crime and I'd appeal to anyone who can help to contact police."
Police would like to hear from anyone who may have noticed anything suspicious in the area at the time or from anyone with information about the robbers.
Call DC Kevin Maskell at Great Yarmouth Police Station on 101 or call Crimestoppers anonymously on 0800 555 111.
4 comments
Pathetic policing, and public awareness. Its time the public dealt with these criminals first hand.
Tuesday, September 18, 2012
Obviously these aren't the types of burglaries that were 20 percent down as reported in the EDP as of Sept 3rd. Any wonder why crime statistics are believed only sceptically?
Tuesday, September 18, 2012
Hmmm,both black males aged 17-18.
Tuesday, September 18, 2012
Muggers on bikes?? Norfolk's going like the rest of Britain....into hell on a handcart.
Tuesday, September 18, 2012
The views expressed in the above comments do not necessarily reflect the views of this site


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Bobby Rodgers
Todd Royval
Bryan Moody
Phil Headden
Scot Churchwell
David Woosley
Tim Commers
Scott Crider
Barry Vradenburgh
Rebecca Shelton
This story is featured in today's TimesFreePress newscast.
Ten white Chattanooga police officers soon may share $725,000 if City Council members today approve settling a 7-year-old racial discrimination lawsuit.
The council is set to vote on a resolution to pay Chattanooga police Lt. Bobby Rodgers and nine other officers the settlement. The resolution aims to avoid a trial over claims that the police department in 2005 refused to promote white officers. The officers claimed that then-Police Chief Steve Parks imposed a promotion freeze but temporarily lifted the ban to elevate a black officer from lieutenant to captain.
According to the lawsuit, the promotion freeze came about after members of the council criticized the department for a perceived lack of ranking black officers.
Rodgers had been with the department for more than 20 years. In his lawsuit, he claimed he had "consistently received outstanding performance reviews and commendations" and met all requirements for promotion from sergeant to lieutenant. In March 2005, according to the suit, there was a lieutenant vacancy when Officer Tracy Arnold, who is black, was promoted to captain. But when Rodgers filed for promotion to fill the spot, he was denied by Parks because of the promotion freeze. The nine other officers, who later joined the lawsuit, had similar claims. At the time of the lawsuit, their experience in the police department ranged from nine to 26 years.
A motion hearing for the case was continued Monday in Hamilton County Circuit Court, "in anticipation of the case being approved [by the council]," according to Steven Dobson, the lawyer representing the 10 officers. If the council doesn't approve the settlement, a motion hearing will be held Aug. 5, according to court records.
Dobson said Monday most of his clients eventually were promoted, and some have retired, but that doesn't absolve the city of discrimination the officers endured previously.
Dobson declined to comment on how the $725,000 settlement would be split if approved, but the initial suit requested that each officer be awarded back pay they would have received had they been initially promoted.
Since the lawsuit, the police department changed its promotion procedure. The old way ranked promotion candidates according to evaluation scores, and the chief of police only promoted from a pool of the top six scorers. Council members had said that system prevented minority officers from being promoted, according to the lawsuit.
Now the chief of police may promote from an alphabetical list of the top 20 scores on a full evaluation.
Council Chairman Yusuf Hakeem was one of the council members pushing for changes that would lead to more black promotions at the time. But he said Monday the "top 20 system" is fair and equitable -- and the case has been "lingering for some time."
"When you look at tests, there are cultural differences and to some degree some biases. I think this levels the playing field," Hakeem said. "I was not the best test-taker, but I think my actions have showed that I am capable."
Hakeem said Monday he would support the settlement.
Dobson said Monday the settlement did not address the promotion procedure or any other request aside from monetary compensation.
"Our suit is really not to redress that; it's really about these plaintiffs," Dobson said. "[The money] would settle the claims of all those plaintiffs."
Lacie Stone, a spokeswoman for Mayor Andy Berke, did not comment specifically on the lawsuit, but she said the city was "in the process of looking at all functions of city government, including workforce development practices across the board." She added the practices Berke is working on are independent of the settlement.
City Attorney Wade Hinton, who has participated in mediation of the case, said Tuesday if the council approves the resolution the issue will be resolved.
Police Chief Bobby Dodd declined immediate comment on the suit and promotion practices at the department Monday, saying he wanted to read the filing completely first.
Contact staff writer Louie Brogdon at firstname.lastname@example.com or 423-757-6481. Follow him on Twitter at @glbrogdoniv.
Louie Brogdon began reporting with the Chattanooga Times Free Press in February 2013. Before he came to the Scenic City, Louie lived on St. Simons Island, Ga. and covered crime, courts, environment and government at the Brunswick News, a 17,000-circulation daily on the Georgia coast. While there, he was awarded for investigative reporting on police discipline and other law enforcement issues by the Georgia Press Association. For the Times Free Press, Louie covers Hamilton County ...
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MEADVILLE - Warren wrestlers D.J. Fehlman and Alex Streich both advanced to the semifinals of Meadville's annual 17-team Tool City Tournament on Friday and several other Dragons remain alive in Saturday morning's wrestlebacks.
The sophomore Fehlman, wrestling at 126 pounds, started the tournament strong with an easy technical fall victory before advancing to the semifinals with a 5-0 decision win over Conneaut's Kyle Lukowich - the No. 2-ranked 132-pounder in D-10 Class AA. Streich, also a sophomore, continued his early season success with a pair of wins at 220, including an injury default victory and a 4-1 decision win.
Both wrestlers remain in the hunt for Tool City championship and will be looking to punch a ticket to the finals in Saturday morning's semifinals.
Despite suffering losses on Friday, Seth Johnson (120 pounds), Jason Peterson (132), Jonah Hordusky (138), Garrett Smead (152), Cody Gunn (160), Chris McNeal (182) and Dan Henry (195) remain alive in the consolation bracket.


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Copyright © 2013 The Authors All rights reserved.
Cell Reports, Volume 3, Issue 2, 328-334, 14 February 2013
Formation of Subnuclear Foci Is a Unique Spatial Behavior of Mating MAPKs during Hyperosmotic Stress
Simon E. Vidal1, 2, 3, David Pincus1, 2, 4, Jacob Stewart-Ornstein1 and Hana El-Samad1, ,
Corresponding author2 These authors contributed equally to this work
3 Present address: Department of Developmental Genetics, Skirball Institute of Biomolecular Medicine, New York University School of Medicine, 540 First Avenue, New York, NY 10016, USA
4 Present address: Whitehead Institute for Biological Research, 9 Cambridge Center, Cambridge, MA 02142, USA
Graphical Abstract
► Yeast mating pathway MAPKs Fus3 and Kss1 form subnuclear foci under hyperosmotic stress ► The transcription factor Ste12 is present in the foci and required for foci formation ► Foci formation requires the kinase activity of the hyperosmotic stress MAPK Hog1 ► Foci may prevent mating pathway activation in response to hyperosmotic stress
- The assembly of signaling components and transcription factors in ordered subcellular structures is increasingly implicated as an important regulatory strategy for modulating the activity of cellular pathways. Here, we document the inducible formation of subnuclear foci formed by two mitogen-activated protein kinases (MAPKs) in Saccharomyces cerevisiae upon hyperosmotic stress. Specifically, we demonstrate that activation of the hyperosmotic stress response pathway induces the mating pathway MAPK Fus3 and the filamentation pathway MAPK Kss1 to form foci in the nucleus that are organized by their shared downstream transcription factor Ste12. Foci formation of colocalized Ste12, Fus3, and Kss1 requires the kinase activity of the hyperosmotic response MAPK Hog1 and correlates with attenuated signaling in the mating pathway. Conversely, activation of the mating pathway prevents foci formation upon subsequent hyperosmotic stress. These results suggest that Hog1-mediated spatial localization of Fus3 and Ste12 into subnuclear foci could contribute to uncoupling the pheromone and osmolarity pathways, which share signaling components, under high-osmolarity conditions.
Mitogen-activated protein kinases (MAPKs), a family of Ser/Thr protein kinases conserved among eukaryotes, are involved in many cellular programs such as proliferation, differentiation, and death (Blenis, 1993; Kim and Choi, 2010; Wagner and Nebreda, 2009). MAPK signaling cascades are organized hierarchically into three-tiered modules. MAPKs are phosphorylated and activated by MAPK kinases (MAP2Ks), which are phosphorylated and activated by MAP2K kinases (MAP3Ks) (Chen and Thorner, 2007). MAP3Ks themselves are activated by interaction with members of small GTPase families and other protein kinases, connecting the MAPK module to cell-surface receptors or other signaling molecules at the plasma membrane. Three main families of MAPKs exist in mammalian species, grouped by their structures and functions: the extracellular signal-regulated protein kinases (ERKs), the p38 MAPKs, and the c-Jun NH2-terminal kinases (JNKs) (Graves et al., 1995; Seger and Krebs, 1995; Wagner and Nebreda, 2009). Transmission of signals to the nucleus and subsequent modulation of the activity of a number of transcription factors and chromatin remodeling proteins is a particularly important role of MAPKs. This transmission is often mediated by the physical translocation of MAPK proteins to the nucleus (Chen et al., 1992; Cohen-Saidon et al., 2009; Khokhlatchev et al., 1998; Plotnikov et al., 2011).
The budding yeast Saccharomyces cerevisiae possesses five genes with homology to mammalian MAPKs (HOG1, SLT2, FUS3, KSS1, and SMK1). These kinases, whose active forms have both cytoplasmic and nuclear targets, are important for inducing the activity of condition-specific transcriptional programs via phosphorylation of transcription factors, and in some cases by serving as transcriptional coactivators (Chen and Thorner, 2007). As in mammalian cells, activation-dependent MAPK nuclear translocation is conserved in S. cerevisiae and has been directly observed for Hog1, the MAPK activated in response to hyperosmotic stress (Ferrigno et al., 1998; Mettetal et al., 2008; Muzzey et al., 2009). Nuclear translocation of Fus3, the pheromone pathway MAPK, and Kss1, the filamentation pathway MAPK, has also been observed (Blackwell et al., 2003, Blackwell et al., 2007; Chen et al., 2010; Maeder et al., 2007; Slaughter et al., 2007).
Hog1 is instrumental for the specific and faithful activation of the hyperosmotic-stress-responsive genes (O'Rourke and Herskowitz, 1998; Saito and Tatebayashi, 2004). Despite the fact that the hyperosmotic stress pathway shares the same MAP3K (Ste11) with the pheromone response and filamentation pathways, hyperosmotic stress fails to activate pheromone- or filamentation-responsive genes (O'Rourke and Herskowitz, 1998; Schwartz and Madhani, 2004; Shock et al., 2009). The remarkable ability of hyperosmotic stress to exclusively activate the hyperosmotic response requires Hog1 kinase activity and has recently been attributed to Hog1-mediated feedback control, potentially through the adaptor protein Ste50 and the kinase Rck2 (Nagiec and Dohlman, 2012). This model, however, does not fully explain the observation that hyperosmotic stress activates Fus3 and Kss1, yet the target genes of the pheromone and filamentation pathways remain off (Shock et al., 2009). As a result, some further insulation at the level of the transcription factors downstream of Fus3 and Kss1 might still occur.
In addition to simply binding to regulatory sites to control gene expression, transcription factors have been shown to organize subnuclear structures with varied functions (Brickner et al., 2012; McCullagh et al., 2010; Sutherland and Bickmore, 2009). In metazoan cells, many subnuclear foci containing transcription factors have been described, such as nuclear stress bodies, histone locus bodies, and polycomb bodies (Mao et al., 2011; Nizami et al., 2010). Rather than being nonfunctional aggregates, these structures within the nucleus implement important layers of regulation.
Here, we show that activation of Hog1 by hyperosmotic stress induces the formation of subnuclear foci containing the transcription factor Ste12 and the MAPKs of the mating and filamentation pathway. Our results are consistent with a model in which these subnuclear structures prevent pheromone and filamentation pathway target gene activation in response to hyperosmotic stress. This work uncovers a previously undocumented spatial behavior of the MAPK proteins and suggests that transcription-factor-mediated sequestration of signaling components could serve as a general mechanism to modulate the activity of signaling pathways.
Fus3 and Kss1 Are Unique among MAPK Proteins in Their Ability to Form Foci under High-Osmolarity Stress
To gain insight into the regulation of localization of S. cerevisiae MAP kinases, we fused yellow fluorescent protein (YFP) to the C termini of Fus3, Kss1, Hog1, and Slt2 and imaged these strains under a panel of metabolic and stress conditions. In vegetatively growing cells, Fus3 and Kss1 were enriched in the nucleus. Slt2 had a reduced nuclear signal compared to Fus3 and Kss1, while Hog1 was mostly cytoplasmic (Figure 1A). In cells treated with 1 μM α factor, none of the four kinases changed their localization until Fus3 partially redistributed from the nucleus to the emerging shmoo tip after 1 hr of treatment (Figure S1A), consistent with previous data (Slaughter et al., 2007).
By contrast, addition of the osmotic stressor sorbitol (0.5 M) triggered the redistribution of Hog1, Fus3, and Kss1. Hog1 transiently localized to the nucleus, within 15 min of treatment and returned to the cytoplasm within 30 min (Figure 1A). Strikingly, following sorbitol treatment, Fus3 and Kss1 appeared in small puncta in approximately half the cells in a given confocal microscope section (Figure 1A). Each cell in which the puncta were visible contained one to three discrete foci 15 min after sorbitol addition, and these foci dispersed within 30 min; Fus3 and Kss1 returned to their prestress diffuse localization in all cells 30 min after sorbitol addition (Figure 1A).
Fus3 also formed subnuclear foci under other hyperosmotic stress conditions such as 0.5 M NaCl and 0.8 M glucose (Figure S1B). Importantly, these subnuclear foci did not form under other stress conditions such as hypoosmotic shock (50% H2O), heat shock (40°C), oxidative stress (0.5% H202), cell wall stress (100 μg/ml zymolyase), ER stress (5 mM DTT), or Tor inhibition (20 mM caffeine) (Figure S1C). These data strongly suggest that the Fus3 and Kss1 foci are a specific phenotype associated with high-osmolarity stress.
Fus3/Kss1 Foci Formation under Hyperosmotic Stress Is Dependent on the Pheromone Response Transcription Factor Ste12
We next investigated whether Fus3 and Kss1-containing foci were the same spatial structure. To do so, we measured the localization of Fus3-YFP and Kss1-mKate2 coexpressed in the same cell upon stimulation with sorbitol. Under these conditions, Fus3-YFP and Kss1-mKate2 colocalized in nuclear foci (Figure 1B), indicating that Fus3 and Kss1 are both contained within the same subnuclear structures.
The presence of nuclear foci involving the pheromone pathway transcription factor Ste12 has previously been documented. In strains deleted for DIG1, a gene encoding a negative regulator of Ste12, Ste12 molecules localize into discrete subnuclear foci (McCullagh et al., 2010). Because of the propensity of Ste12 to form these structures in dig1Δ cells, we hypothesized that Ste12 molecules might also be components of the Fus3/Kss1 foci. To test this possibility, we monitored the localization of Fus3-mKate2 and Ste12-YFP coexpressed in the same cells. Upon treatment with 0.5 M sorbitol, Ste12 indeed formed nuclear foci. Importantly, Fus3 colocalized with Ste12 in these foci (Figure 1C). Moreover, just as Ste12 is constitutively localized in foci in dig1Δ cells, Fus3 also appeared in constitutive foci in the absence of sorbitol in dig1Δ cells (Figure S1D). Furthermore, both Dig1 and its paralog Dig2 were present in sorbitol-induced foci in wild-type cells (Figure S1E). In summary, these data indicate that the MAPKs Fus3 and Kss1, along with their shared transcription factor Ste12 and its negative regulators Dig1 and Dig2, are components of hyperosmotic-stress-induced subnuclear structures.
To determine which components are required for the emergence of these structures, we monitored foci formation upon sorbitol addition in strains lacking FUS3, KSS1, or STE12. Interestingly, neither individual deletions of FUS3 or KSS1, nor the FUS3/KSS1 double deletion impacted the formation of Ste12 foci (Figure 1D). By contrast, Fus3 and Kss1 failed to form foci in ste12Δ cells under osmotic stress conditions (Figure 1D).
Since Ste12, a transcription factor, is a crucial component for the formation of these foci, we sought to ask whether DNA also associated with these foci. To test this possibility, we performed ChIP-PCR experiments using Ste12-YFP as bait in untreated, pheromone-treated, or sorbitol-treated cells (Figure S1F). In untreated cells, we found that Ste12 associated with pheromone target gene promoters (AGA1 and FUS1), but not with osmo-stress target gene promoters (HOR2 and GPD1) or metabolic gene promoters (PGK1 and TDH3). We did not observe Ste12 binding to osmo-stress gene promoters or metabolic gene promoters in any condition. Notably, we found an increase in Ste12 binding to pheromone target promoters upon pheromone treatment, but we did not observe any depletion of Ste12 from the pheromone target gene promoters upon sorbitol treatment (Figure S1G). These data indicate that Ste12 remains specifically bound to its target gene promoters while simultaneously localizing to subnuclear foci.
Genomic loci have been reported to localize to the nuclear membrane both for activation and repression (Brickner and Walter, 2004; Green et al., 2012). Since the hyperosmotic-stress-induced foci are associated with DNA, we next asked if the foci localize to the nuclear periphery. To this end, we monitored Fus3-YFP in cells in which the nuclear pore component Nup49 was fused to mCherry. After addition of sorbitol, we observed that Fus3 foci indeed localized with or near Nup49 (Figure 1E). Taken together, these data demonstrate that the common transcription factor Ste12 is necessary to organize the MAPKs Fus3 and Kss1 into the subnuclear foci induced by high-osmolarity stress, and that these foci are associated with DNA and are localized to the nuclear periphery.
Hog1 Kinase Activity Is Required for Formation of Fus3/Kss1/Ste12 Foci in Conditions of Hyperosmotic Stress
Under high-osmolarity conditions, the effector MAPK Hog1 is phosphorylated by the MAP2K Pbs2 and translocates from the cytoplasm to the nucleus where it plays transcriptional and posttranslational roles in the hyperosmotic stress response (Ferrigno et al., 1998; Mettetal et al., 2008). Therefore, Hog1 is a prime candidate for a regulatory role in Fus3/Kss1/Ste12 foci formation. To address this notion, we first investigated whether Hog1 residence in the nucleus correlated with Fus3/Kss1/Ste12 foci formation. Fus3 and Ste12 foci form and dissipate with identical kinetics (Figures S2A and S2B), so we chose to simultaneously monitor Fus3-YFP and Hog1-mKate2 in the same cell. The kinetics of Fus3 foci formation and dissolution correlated with Hog1 nuclear residence (Figures 2A and 2B). Hog1 began to translocate to the nucleus within 2 min of 0.5 M sorbitol, and Fus3 began to form foci within 3 min. Both Hog1 nuclear enrichment and Fus3 foci formation achieved their peaks between 5 and 10 min after sorbitol addition. Within 15 min, Fus3 foci began to dissipate while Hog1 persisted longer in the nucleus, beginning to relocalize to the cytoplasm only after 20 min (Figures 2A and 2B). Due to our imaging technique, we were not able to observe Fus3 foci in all cells, likely due to the foci falling above or below the confocal plane. Nonetheless, the strong correlation between Fus3 foci and Hog1 nuclear residency held across a range of osmotic stress magnitudes that produced different but correlated Hog1 and Fus3 dynamics (Figure S2C). Moreover, Fus3, Kss1, and Hog1 are phosphorylated and dephosphorylated with similar kinetics, matching the kinetics of foci formation and Hog1 nuclear translocation (Figure S2D).
To directly test if Hog1 activity is required for foci formation, we constructed an analog-sensitive allele (hog1-as) whose kinase activity is susceptible to inhibition by the cell-permeable ATP analog 1-NM-PP1 (Westfall and Thorner, 2006). In this mutant, and by adding 1-NM-PP1, pheromone genes are expressed under high-osmolarity stress (Westfall and Thorner, 2006). The hog1-as allele was inserted into the genome of a hog1Δ strain under the control of the native HOG1 promoter. In hog1-as cells, 1-NM-PP1 alone had no effect on Fus3 localization. Moreover, in hog1-as cells, Fus3 formed nuclear foci at a frequency comparable to that of wild-type cells when treated with 0.5 M sorbitol alone (Figures 2C and 2D). By contrast, less than 10% of cells pretreated with 1 μM 1-NM-PP1 formed foci upon addition of 0.5 M sorbitol (Figures 2C and 2D). These data demonstrate that the kinase activity of Hog1 is required for Fus3 foci formation under high-osmolarity stress.
Preactivation of the Pheromone Pathway Inhibits the Formation of Fus3 Foci
The experiments described above show that Ste12 and Fus3, components of the pheromone response pathway, are recruited into foci in high-osmolarity conditions and that Hog1 kinase activity is required for this process to proceed. Despite the fact that the hyperosmotic stress pathway shares the same MAP3K (Ste11) with the pheromone pathway, hyperosmotic stress fails to activate pheromone-responsive genes (O'Rourke and Herskowitz, 1998; Schwartz and Madhani, 2004; Shock et al., 2009). Therefore, foci formation could constitute a spatial mechanism for attenuating signaling in the pheromone pathway when cells are exposed to high osmolarity. In this case, the presence of foci should diminish the induction of pheromone target genes. Conversely, conditions where these foci are absent should coincide with unattenuated pheromone signaling output.
To test the notion that the Fus3/Kss1/Ste12 foci represent a spatial mechanism for attenuating pheromone pathway output during hyperosmotic stress, we performed a series of experiments in which we treated cells with both α factor and sorbitol. We hypothesized that if cells treated simultaneously with sorbitol and pheromone prioritize the hyperosmotic stress response, then these cells would form foci that attenuate signaling in the pheromone pathway. On the other hand, we reasoned that pretreatment of cells with α factor would establish active signaling through the pheromone MAPK cascade that would preclude the formation of foci. The absence of foci in this situation should then correlate with unattenuated signaling and induction of pheromone-responsive gene expression.
To test these hypotheses, we first used confocal microscopy to monitor Fus3-YFP cells treated with both 1 μM α factor and 0.5 M sorbitol either simultaneously or by pretreating the cells with α factor for 10 min before sorbitol addition. When cells were exposed to α factor and sorbitol simultaneously, Fus3 formed foci to the same level as in cells treated with sorbitol alone (Figure 3A). However, when cells were preincubated in 1 μM α factor for 10 min, addition of 0.5 M sorbitol failed to induce Fus3 foci (Figure 3A). Deletion of STE7, the MAP2K upstream of Fus3, rescued Fus3 foci formation in cells pretreated with pheromone and subsequently treated with sorbitol, showing that signaling through the pheromone MAPK cascade is required to prevent foci formation (Figure 3A).
We next monitored PAGA1-YFP, a transcriptional reporter of mating pathway activity, by flow cytometry to measure the output of the pheromone pathway in cells treated with both 1 μM α factor and nine different doses of sorbitol, either simultaneously or with pheromone pretreatment. When cells were simultaneously treated with 1 μM pheromone and sorbitol—conditions in which Fus3 foci form—mating pathway output was diminished in proportion to the dose of sorbitol, consistent with previous data (Nagiec and Dohlman, 2012) (Figure 3B). However, in cells pretreated with 1 μM α factor for 10 min before sorbitol addition—conditions that abolished Fus3 foci formation—PAGA1-YFP exhibited full induction under all sorbitol concentrations (Figure 3C). A control promoter for a metabolic gene that is not pheromone regulated (PTDH3-mCherry) remained constant over time in all conditions, demonstrating that the effects of sorbitol are specific to the pheromone pathway reporter (Figure S3). These data indicate that preactivation of Ste12 by pheromone signaling inhibits the formation of Fus3/Kss1/Ste12 foci upon subsequent hyperosmotic stress and abrogates the hyperosmotic stress inhibition of the pheromone pathway output. These results are consistent with the hypothesis that spatial localization of Fus3, Kss1, and Ste12 into foci could constitute a strategy to isolate the pheromone pathway under high-osmolarity conditions.
The activities of many signaling proteins are regulated by their physical proximity and access to substrates. A common form of this regulation in eukaryotes is controlling access of transcription factors to DNA by regulating import into the nucleus (Cyert, 2001). An extension to this emerging regulatory model is the control of subnuclear localization of transcription factors and genes to the nuclear periphery, nucleoli, or foci (Brickner et al., 2012; Mao et al., 2011; Nizami et al., 2010). Such foci of colocalized transcription factors and coordinately regulated DNA have been observed in metazoans and fungi and have been suggested to represent transcriptional factories where common transcriptional machinery can be marshaled and therefore modulate gene expression of the localized genes (Sutherland and Bickmore, 2009).
Recent work on the Ste12 transcription factor in yeast suggests that cells prevent the colocalization of genes regulated by this transcription factor through the use of a dedicated repressor, Dig1. The absence of Dig1 results in clustering of Ste12 and its target gene promoters, and a concomitant increase in gene expression noise (McCullagh et al., 2010). Since Ste12 clustering is not observed in wild-type cells upon activation by pheromone stimulation, this clustering is likely to play a role other than activation of gene expression.
Our present work reveals that Ste12 also forms subnuclear foci physiologically in wild-type cells in response to hyperosmotic stress. In these foci, Ste12 colocalizes with the upstream MAPKs Kss1 and Fus3 and the negative regulators Dig1 and Dig2. Whether and to what extent the formation of Ste12 foci in the two conditions, dig1Δ cells and cells experiencing hyperosmotic stress, reflects the same underlying mechanism remains to be determined. The most parsimonious model is that Ste12 clustering is a controlled process during hyperosmotic stress, but gets unspecifically triggered in the absence of Dig1. The fact that foci formation of colocalized Ste12, Fus3, and Kss1 was only observed under hyperosmotic stress and required the kinase activity of the osmotic stress MAPK Hog1 supports the hypothesis that these foci represent a specific response to osmotic shock rather than a general response to cellular damage.
Hyperosmotic stress poses a unique challenge to the pheromone response and filamentation pathways that share a common MAP3K, Ste11. Both the MAP2K Ste7 and the MAPKs Fus3 and Kss1 become phosphorylated during high-osmolarity stress (Shock et al., 2009). Yet, downstream target genes of the pheromone and filamentation pathways remain inactive during osmotic stress. Hog1 kinase activity-mediated clustering of Ste12, Fus3, and Kss1 into subnuclear foci may represent a spatial mechanism that prevents the inappropriate activation of pheromone and filamentation genes despite the activation of their MAPKs by osmotic stress. Repression of signaling by foci formation has recently been shown to occur in the TOR pathway in yeast upon heat shock, suggesting that attenuation by spatial sequestration might be a general regulatory mechanism (Takahara and Maeda, 2012).
Despite the tight correlation of Ste12/Fus3/Kss1 foci formation with Hog1 activation and the inhibition of pheromone target gene expression, much work is still needed to elucidate the mechanisms of Ste12 foci formation and demonstrate a causal relationship between these foci and crosstalk inhibition between the hyperosmolarity and pheromone response pathways. Nonetheless, a tantalizing model suggested by our data is one in which Fus3 and Kss1 are activated by crosstalk upon osmotic stress, while the activation of downstream target genes is suppressed by sequestration of these kinases into foci. This method of inhibition would be complementary to Hog1-directed repression of upstream pheromone pathway elements such as Ste50. Both mechanisms would collaborate to prevent activation of the pheromone inducible target genes by hyperosmotic stress, which is crucial given the toxic effects of costimulation of pheromone and osmotic pathways (Patterson et al., 2010). This physical sequestration of kinases into subnuclear structures organized by their downstream transcription factors provides the overlapping pheromone, filamentation, and osmotic stress signaling pathways with a rapid and reversible way to prevent inappropriate signaling.
Experimental Procedures
A detailed description of the experiments is included in the Extended Experimental Procedures. Briefly, standard procedure for growth, maintenance, and transformation of yeast and bacteria and for the manipulation of DNA were used throughout the study (Document S1. Tables S1–S3 and Document S2. Article plus Supplemental Information). Subcellular localization of fluorescent fusion proteins was observed by spinning disc confocal microscopy. Reporter fluorescence was measured by flow cytometry. Western blots were visualized via infrared fluorescence using the Li-Cor imaging system. ChIP samples were assayed for enrichment by PCR (Document S1. Tables S1–S3).
Strains, Plasmids, and Growth Conditions
All yeast strains used are derived from W303a and are described in supplementary material (Document S1. Tables S1–S3). Standard molecular biology techniques were used to construct the transcriptional reporter (PAga1-YFP) and the Hog1 analog sensitive allele (Document S1. Tables S1–S3). Point mutations were generated with the QuikChange site-directed mutagenesis kit (Stratagene) according to the manufacturer's directions. Protein fusions were constructed by homologous insertion of a YFP or RFP protein (mKate2) at the 3′ end of the open reading frame with a His3 or Ura3 marker immediately downstream. All experiments were conducted in synthetic media (2 × SDC: yeast nitrogen base, glucose, complete amino acids). Plasmid-transformed cells were grown in synthetic medium lacking the appropriate nutrient.
Addition of Stress Inputs
In order to activate the high-osmolarity pathway, cells were grown to OD600 ∼0.2, followed by addition of appropriate quantities of Sorbitol (2M stock, Sigma), glucose (50% solution, Sigma), or NaCl (5M stock, Sigma). For the hog1-as mutant, cells were treated with 1 μM 1-NM-PP1 (gift from the Shokat lab) for 20 min to completely inhibit the kinase, followed by addition of the osmotic stressor. Stocks of α-factor pheromone (Sigma) were resuspended at 600 μM, stored at −20°C and added to the cells at a final concentration of 1 μM. For the foci formation assays using other stressors, we exposed cells to hypo-osmotic shock (50% H2O), oxidative stress (0.5% H202), cell wall stress (100 μg/ml zymolyase), ER stress (5 mM DTT) and Tor inhibition (20 mM caffeine). To heat shock cells, 40 μl of 50°C media was added to 40 μl of cells at 30°C; 40μl of media was replaced with 40° media every 10 min. Images were taken every 10 min by 30 min.
Cells harboring the gene fusions were grown at 30°C in 2 × SDC to mid log phase, diluted to OD600∼ 0.2, gently sonicated, and 80 μl added to 96 well glass bottom plates coated with concanavalin-A. Cells were allowed to settle for 30 min and images were taken using a using a 100X 1.49 NA Nikon lens, with a Yokogawa CSU-X MI spinning disc confocal on a Nikon Ti-2000 inverted microscope. GFP and YFP were excited with a 491 nm laser line, and emission was collected with a 525/50nm filter set (Semrock); RFP was excited with a 561 nm laser line and emission collected by a 595/50nm filter set (Semrock). To induce hyper-osmotic stress, we added sorbitol, glucose or NaCl to final concentrations of 0.5, 0.8 and 0.5M, respectively. For each time point we obtained images of three fields of cells, collecting a total of 30–60 cells per time point. Experiments were performed 3 or more times. Images were analyzed semi-manually in ImageJ, statistics were computed with Excel.
Transcriptional Reporter and Flow Cytometry
Cells were grown to saturation in 96-shallow-well plates (Costar) and then diluted into fresh media to an OD of ∼ 0.05 and subsequently grown at 30°C on orbital shakers (Elim) at 900 rpm for 12 hr to an OD of ∼ 0.2 before measurement. Single cell flouresence was measured on a BD LSR-II equipped with a high throughput sampler, YFP was excited with a 488 nm laser and emission collected through a 530/30nm filter (chroma). Flow cytometry data was analyzed in FloJo. Error bars were calculated using the standard error of the mean for 3 biological replicates.
Protein Extraction and Western Blotting
Total cellular protein was extracted from yeast cells as previously described (Yaffe and Schatz, 1984). Samples were run on BioRad ready-gels (4%–15% acrylamide, Tris/glycine/SDS) for 90 min at 35 mA. The proteins were subsequently transferred to Millipore Immobilon PVDF membranes at 220 mA for 45 min. Blots were blocked in 1% casein in TBS (10 mMTris, 150 mMNaCl) for 30 min, followed by incubation with primary antibodies overnight. The rabbit polyclonal anti-Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) (cell signaling) and the mouse monoclonal anti-body Phospho-p38 MAPK (Thr180/Tyr182)(cell signaling) were used at 1:1000 dilution. The next morning, the blots were washed 3 × for 10 min with TBS, and then incubated with Li-Cor fluorescently-coupled secondary antibodies, goat anti-mouse and rabbit 680 and 800, at 1:10,000 dilution for 30 min. Blots were again washed 3 × for 10 min with TBS, scanned with the Li-Cor infrared scanner, and processed with the Odyssey software package.
Chromatin Immunoprecipitation
A saturated culture of Ste12-YFP cells was diluted into fresh YPD media (50ml) to an OD ∼0.02 and grown to an OD ∼ 0.3-0.4. α-factor and sorbitol were added to a final concentration of 1 μM and 0.5 M and incubated for 30 and 8 min, respectively. 1.4 ml of 37% formaldehyde was added to each flask and incubated at RT for 15 min with occasional swirling. 2.5 ml of 2.5 M glycine was added and incubated at RT for 5 min with occasional swirling to quench the formaldehyde. Cells were centrifuged at 2000 rpm for 5 min at 4°C. Pellet was washed 2X with 30 ml TBS and transferred to a 2 ml screw cap tube. Cells were centrifuged at 14000 rpm for 5 min at 4°C. Supernatant was aspirated and tubes were frozen in liquid nitrogen. Cells were resuspended with 500 μl Lysis Buffer (50mM HEPES-KOH pH 7.5, 140mM NaCL, 1mM EDTA, 1% Triton X-100, 0.1% Na deoxycholate and protease inhibitor cocktail) and glass beads were added to each tube. Cells were bead-beaten at maximum setting 5x1 min with 2 min rests on ice. The bottom of the tubes were punctured using a 23G1 needle and placed them in 5 ml snap cap tubes. Tubes were centrifuged at 1000 rpm for 3 min at RT. The collected lysate and pellet was resuspended and transferred to a new tube. Tubes were centrifuged at 8000 rpm for 10 min at 4°C. Pellet was resuspended in 500 ul lysis buffer and sonicated in a Bioruptor sonicator bath for 2x15 min using intervals of 30 s on and 1 min off. Tubes were centrifuged at 14,000 rpm for 10 min at 4°C. Supernant (WCE) was transferred to a new tube and 50 ul was taking for PCR analysis. Anti-GFP antibody coupled to agarose beads (chromotek) was added to each WCE and nutated for 2 hr at 4°. Tubes were centrifuge 30-60 s 500 rpm at RT and aspirate supernatant. Beads were washed 2x5 min with 1 ml lysis buffer, 2x5 min with 1 ml high salt lysis buffer (50mM HEPES-KOH pH 7.5, 500mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% NaDOC), 2x5 min with 1 ml wash buffer (10mM Tris-Cl pH 8.0, 0.25M LiCl, 0.5% NP-40, 0.5% NaDOC, 1mM EDTA), 1x5 min with 1 ml TE, pH 8.0. Finally 100 ul of elution buffer (50mM Tris-Cl pH 8.0, 10mM EDTA, 1% SDS) was added to each tube and incubated for 15 min at 65°. Tubes were centrifuged at 14,000 rpm for 15 min. Supernant was transfered to new eppendorf and 150 ul TE/0.67% SDS (with Proteinase K) was added to the remaining beads. Tubes were centrifuged at 14,000 rpm for 5 min and supernatant was transferred to the first elutate (final volume 250 ul). 200 ul of TE/1% SDS (with PK) was added to 50 ul WCE aliquots. Chromatin immunoprecipitation (ChIP) and WCE were incubated at 65° O/N. Finally 55μL 4M LiCl and 500 μl Phenol/Chloroform/Isoamyl alcohol were added to each tube. Tubes were vortexed, centrifuged and the aqueous layer was removed to a fresh tube. 1 ml of EtOH was added to each tube and incubated at −80°C for 2hrs. Tubes were centrifuged at full speed for 30 min at 4°C. Pellet was washed with 1 ml 75% EtOH and centrifuged at full speed for 5 min at 4°C. Pellet was air-dried and resuspended in 50 μl TE buffer. For the PCR, WCE were diluted 1/100 and 0.5 ul, 3 ul and 10 ul were used as input. The IP was not diluted and 1ul was used. Bands from the gel were assigned values in ImageJ, and using the standard curve from the dilution series of WCE, IP bands were quantified as a percent of the input DNA. Primers that were used for PCR are shown in Document S1. Tables S1–S3.
We would like to thank Noah Helman and Wendell Lim for the pNH604 and pNH605 vectors, and Chao Zhang and Kevan Shokat for the ATP analog 1-NM-PP1. We are grateful to Hiten Madhani and Ignacio Zuleta for valuable scientific discussions and to members of the El-Samad laboratory for help and advice during the course of this work. The confocal microscope was provided by the UCSF-based Consortium for Frontotemporal Dementia Research housed at the J. David Gladstone Institutes. This work was funded by the NIGMS System Biology Center (P50 GM081879). S.E.V., D.P., J.S.-O., and H.E.-S. designed the experiments, analyzed the data, and wrote the manuscript. S.E.V., D.P., and J.S.-O. performed the experiments.
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Publication Information
Received: July 10, 2012
Revised: October 9, 2012
Accepted: January 16, 2013
Published: February 14, 2013


text:
Green gambling, but don't let this guy run your numbers
Thomas Friedman visited a wind farm near the East Asian gambling capital, Macao. But his rhetoric outsizes his quantitative skills in setting up another "dichotomy" in a "flat" world.
The column is a dizzying and logically disjointed ramble through some well-worn tropes on China's economy that have developed during the media's concurrent green awakening and Olympic China craze in recent months.
This is not so surprising from a columnist specialists love to lambaste, but this opening left me more confused than usual:
[T]he Chinese engineers showed me their control room, which has a … Read more


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Volume Two of The Seven-Petaled Shield
Deborah J. Ross - Author
ISBN 9780756409203 | 464 pages | 03 Dec 2013 | DAW | 6.49 x 4.29in | 18 - AND UP
Summary of Shannivar
Summary of Shannivar
Reviews for Shannivar
An Excerpt from Shannivar
Shannivar is a young warrior, skilled in horsemanship and archery and raised by her uncle, the clan chieftain. When all the other young warrior-women have set aside their bows and arrows for husbands, she still dreams of glory. Desperate to resist the imperialistic arm of the nearby growing empire, all of the clans of her nation are gathering to propose an alliance with each other and nearby, still-independent countries. As Shannivar and her fellow delegates travel to the meeting, they encounter a stranger, Zevaron, heir to the magical Seven-Petaled Shield. Zevaron also seeks to halt the empire's expansion on behalf of his own fallen lost nation and joins forces with Shannivar. Shannivar has never met anyone like Zevaron, with his aura of power and quiet assurance. Soon the clan gathering is ready, except for the mysterious absence of the clan from the far north. When the ragged band of the missing delegates finally arrive, they tell strange and ominous stories of wolves running mad, reindeer turning cannibal, and man-sized, upright lizards, called stone-drakes. Zevaron, bestowed with the ancient central petal of the mystical Seven-Petaled Shield, feels a great emanation of danger from the north upon learning this news. Together, he and Shannivar set out on a journey to discover the source of these dangers—a quest even the prophets believe will fail. "Rejoice.... Ross's literary strength [is] her sensitivity to human needs and talents."—Publisher's Weekly "Deborah has contrived to express the rich tapestry of the characters and backdrop of Darkover in a way that would make Marion proud."—ConNations "Ross succeeds in keeping [Darkover] true to Bradley's style."—Booklist "Fans will be pleased, and those new to the world of Darkover have a treat in store."—VOYA |
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