Cells were lysed in RIPA buffer (150 mM NaCl, 0.5% sodium deoxycholate, 1% NP40, 0.1% SDS, 50 mM Tris-HCl [pH 8.0]) containing protease inhibitor and phosphatase inhibitor cocktails (Roche). Harvested cell extracts were separated by 10% SDS-PAGE and transferred to PVDF membranes, which were reacted with primary antibody, and then horseradish peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratory) and visualized with an enhance chemiluminescence system (Thermo). Images were acquired by a digital image system (UVP or Fujifilm).