HEK293 cells were obtained from the American Type Culture Collection (ATCC) and cultured under standard conditions recommended by the ATCC. HEK293 cells were cultured on 60-mm dishes to 40 to 50% confluence, treated with tunicamycin for 72 hours at the indicated concentrations to induce ER stress, and harvested for immunoblotting with anti-CDK4 antibody.
For immunoprecipitation with anti-CDK4, HEK293 Ctrl or shCHIP cells were incubated with tunicamycin (1 μg/ml) for 24 hours and then treated with 10 μM MG132 for 1.5 hours. The cells were lysed by sonication in RIPA lysis buffer as described previously (13 (link)). Four milligrams of protein lysates was incubated with 20 μl of anti-CDK4 agarose (#sc-260, Santa Cruz Biotechnology) overnight at 4°C. The beads were washed with RIPA buffer three times, and 60 μl of 1× SDS-PAGE loading buffer was added. The sample was boiled for 5 min. Half of the immunoprecipitates or 50 μg of the total protein lysate was loaded for SDS-PAGE and assayed by Western blotting. The membranes were probed with anti-HSP70 (SC-24, Santa Cruz Biotechnology), anti-HSP90 (SC-13119, Santa Cruz Biotechnology), anti-CDC37 (SC-13129, Santa Cruz Biotechnology), anti-CHIP (SC-133066, Santa Cruz Biotechnology), and anti–α-tubulin antibody (T6199, Sigma-Aldrich).
For immunoprecipitation with anti-CDK4, HEK293 Ctrl or shCHIP cells were incubated with tunicamycin (1 μg/ml) for 24 hours and then treated with 10 μM MG132 for 1.5 hours. The cells were lysed by sonication in RIPA lysis buffer as described previously (13 (link)). Four milligrams of protein lysates was incubated with 20 μl of anti-CDK4 agarose (#sc-260, Santa Cruz Biotechnology) overnight at 4°C. The beads were washed with RIPA buffer three times, and 60 μl of 1× SDS-PAGE loading buffer was added. The sample was boiled for 5 min. Half of the immunoprecipitates or 50 μg of the total protein lysate was loaded for SDS-PAGE and assayed by Western blotting. The membranes were probed with anti-HSP70 (SC-24, Santa Cruz Biotechnology), anti-HSP90 (SC-13119, Santa Cruz Biotechnology), anti-CDC37 (SC-13129, Santa Cruz Biotechnology), anti-CHIP (SC-133066, Santa Cruz Biotechnology), and anti–α-tubulin antibody (T6199, Sigma-Aldrich).