Immunoprecipitation and immunoblot
The cells were harvested and lysed on ice by 0.4 ml of the modified RIPA buffer (50 mM Tris-HCl [pH 7.4], 1% NP-40, 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 1 mM NaF) supplemented with 1 × protease inhibitor cocktail (Roche) and 1 × phosphatase inhibitor cocktail set I (EMD Biosciences) for 30 min. The lysates were centrifuged at 10,000 × g at 4 °C for 10 min to remove insoluble material. After normalizing protein concentrations, lysates were subjected to immunoprecipitation by adding 10 mg/ml appropriate antibody plus 30 ml of protein G-agarose (Roche), and rotated for at least 2 h at 4°C. The precipitates were washed at least five times with cold lysis buffer followed by separation by SDS-PAGE under reduced and denaturing conditions. Nitrocellulose membranes were blocked in 5 % nonfat milk in 0.1 % PBS-Tween 20 (PBS-T), probed with specific antibodies as described previously6. For immunoblotting of phosphorylated proteins, gels were transferred to methanol-treated polyvinylidene chloride membranes, retreated with methanol, and dried for 30 min. Blots were blocked in 5 % bovine serum albumin in 0.1 % Tris buffered saline-Tween 20 (TBS-T), and probed with specific antibodies as described previously46. Bands were imaged by the Super Signaling system (Pierce) according to the manufacturer's instructions.