Western blot

Cells were rinsed twice in PBS and lysed by radio immunoprecipitation assay buffer containing protease inhibitors and phosphatase inhibitors. The protein concentration was determined by using Bradford method. Proteins from total lysates were subjected to 5%–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred onto polyvinylidene difluoride membrane. The membrane was blocked with 5% nonfat milk in the mixture of PBS plus 0.1% Tween 20 (PBST) for 1 h and then incubated overnight with primary antibodies. The next day, after three washes in PBST for 10 min, the membrane was incubated with horseradish peroxidase–labeled second antibodies for 1 h at 37°C. After washing, blots were visualized by enhanced chemiluminescence substrate. Quantification of protein bands was performed by using the Gel-Pro analyzer Version 4 software.