Co-immunoprecipitation (Co-IP) and Western blot analysis

After various treatments, the whole cellular lysates were prepared by harvesting the cells in 1× cell lysis buffer (20 mM HEPES [pH 7.6], 150 mM NaCl, and 0.1% NP40 supplemented with 1 mM β-glycerophosphate, 1 mM Na₃VO₄, 1 mM PMSF, 1 mM NaF, 1 mM benzimedin, and protease inhibitors (protease inhibitor cocktail set III; Calbiochem-Novabiochem, San Diego, CA)^37–39. Approximately 200 μg nuclear extract or 1 mg total protein lysate was precleared with 70 μL of slurry of protein A or G Dynabeads (Upstate Biotechnology) for 2 h at 4 °C. Dynabeads (70 μL) were coated with 2 to 5 μg antibodies at 4 °C overnight. The wash buffer (1× TBS, 0.1% Tween® 20) was used to elute proteins. The immunoprecipitates and whole cell lysates were subjected to Western blotting using established methods^3,37. The Western blot was quantified using the Image J Software from the U.S. National Institutes of Health (NIH).