HEK293 cells and HEK293-PTH1R cells were plated in a 6-well plate at a density of 500,000 cells/well and incubated at 37 °C/5% CO2. At 70-80% confluency, the media was replaced with DMEM (serum-free), and cells were incubated for 1h, following which the cells were incubated with teriparatide (10 nM) for 0, 10, 15, 30, and 45 min. After incubation with teriparatide, the cells were lysed as described above using TBS buffer instead of DPBS. The protein content in the cell lysates was determined, and 15 μg total protein were separated by gel electrophoresis and blotted onto a nitrocellulose membrane as described above (Western Blot). Each sample was blotted in duplicates to probe for total p44/42 MAPK (Erk) and phosphorylated p44/42 MAPK (pErk). After blocking the membranes, they were incubated with Erk (1:1000, Cell Signalling Technology, #4695S) and pErk (1:2000, Cell Signalling Technology #4370S) in 5% BSA blocking buffer. An anti-rabbit (1:5000) secondary antibody was prepared in TBS-T. Antibody staining (including tubulin loading control) and imaging was achieved as described above.