Western Blotting Assay Protocol

Western blotting assays were performed as previously described (73 (link), 74 (link), 75 (link)). Briefly, cells were lysed with ice-cold radioimmunoprecipitation assay lysis buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, and 1% Triton X-100), and the concentrations of the extracted proteins in supernatant were quantified by bicinchoninic acid protein assay kit (Beyotime Biotech). About 30 μg of each protein sample was loaded on SDS-PAGE gel. After the SDS-PAGE electrophoresis, proteins were transferred on a polyvinylidene difluoride membrane, and blocked with 5% skim milk in Tris-buffered saline with Tween-20 (10 mM Tris–HCl, pH 8.0, 150 mM NaCl, and 0.05% Tween-20). Specific proteins were probed with the relevant primary antibodies followed by secondary antibodies. The immunoblots were visualized with ECL plus Chemiluminescence kit (Thermo Fisher Scientific). The antibodies used are listed in Table S4.