Western blot

Western blot analysis was performed as described previously⁸. The cells were lysed in the radioimmunoprecipitation (RIPA) lysis buffer containing protease inhibitor. The protein concentrations were determined using a bicinchoninic acid (BCA; Thermo Fisher Scientific Inc) Protein Assay Kit. Equal amounts of proteins were separated through 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) by electrophoresis, and then samples were transferred onto a polyvinylidene fluoride (PVDF) membrane. After blocked with 5% nonfat dried milk, the PVDF membranes were immunoblotted with specific primary antibodies, respectively. Following extensively washing, the membranes were incubated with corresponding secondary antibodies conjugated with horseradish peroxidase. The immune complexes on the PVDF membrane were detected using an Electrochemiluminescence (ECL) detection system.