Western Blot Analysis of Protein Extracts

Western blot analysis was performed as described previously (19 (link)). Briefly, total protein extract was prepared from the cultured cell lines using radioimmunoprecipitation assay lysis and extraction buffer (Thermo Fisher Scientific, #89900) supplemented with protease inhibitor cocktail (Sigma-Aldrich, #11697498001). The concentration of total protein was quantitated using the Bradford dye-binding method (Bio-Rad, #5000006). Twenty micrograms of protein was loaded and separated by 4 to 15% Mini-PROTEAN TGX precast polyacrylamide gel (Bio-Rad, #4561085) and then transferred to 0.22 μm polyvinylidene fluoride (PVDF) membranes (Millipore, #ISEQ00010). PVDF membranes were blocked with 5% nonfat milk and incubated with specific antibodies for detecting different proteins (see detailed information about antibody information in table S2). After the blot is incubated in enhanced chemiluminescence chromogenic substrate (Millipore, #WBKLS0100), protein bands were detected by the ChemiDoc Touch Imaging System (Bio-Rad), and the signal was quantified using Image Lab software (Bio-Rad).