Western Blot Analysis of Cell Lysates

Cell lysates were collected as previously described.^{38 (link)} Thirty-microgram lysates were separated on 6–12% sodium dodecyl sulfate-polyacrylamide gels and transferred to PVDF membranes (Millipore, Bedford, MA, USA). TBST containing with 5% nonfat milk or bovine serum albumin was used to block nonspecific binding for 2 h at room temperature. Then membranes were incubated with primary antibodies according to the instructions overnight at 44 °C, followed by appropriate HRP-conjugated secondary antibodies (1:5000 dilutions). Signals generated by enhanced chemiluminescence (Millipore) were recorded with a CCD camera (CLINX, Shanghai, USA). Data are representative of at least three independent experiments.