After harvesting, the cells were lysed in 50 mM Tris-HCl (pH 8.0) containing 150 mM NaCl, 1% nonidet P-40, protease inhibitor cocktail, 1 mM phenylmethane sulfonyl fluoride, 25 mM sodium fluoride, and 1 mM sodium orthovanadate. Protein concentrations were determined using BCA Protein Assay Kit (Pierce, Rockford, IL, USA). Subsequently, cellular extracts (45~60 μg) were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane. Non-specific binding was blocked with 5% skim milk in Tris-buffered saline with Tween 20 for 30 min at room temperature. The membranes were then incubated with the primary antibody (1 : 1,000) overnight at 4°C and continually with the secondary antibody (1 : 5,000) at room temperature for 30 min. After incubation, the membranes were washed three times for 10 min each. Proteins were visualized using ECL reagent, and the band intensity was measured using a Chemidoc XRS densitometer system and quantified by Quantity One software (Bio-Rad).