Western blot assay

Cells were lysed in RIPA buffer and the lysates were normalized using a BCA protein assay kit (Beyotime, China). Total proteins were separated by 10–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was blocked in 5% non-fat milk and incubated with the indicated primary antibodies at 4 °C overnight. Then, proteins were detected by incubation with species-specific, peroxidase-conjugated secondary antibodies. The immunoreactive bands were detected using a chemiluminescence kit (Millipore, Plano, TX, USA) and visualized with a KODAK imaging system.