2.7 | Western Blotting
Proteins were extracted using radioimmunoprecipitation assay (RIPA)
lysis buffer (Beyotime Biotechnology, China), separated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to a
polyvinylidene fluoride (PVDF) membrane. PVDF membranes were blocked in
TBST (pH 7.4, containing 0.05% Tween 20 and 5% non-fat milk) for 1.5 h
at room temperature before adding specific primary antibodies.
Subsequently, the PVDF membranes were incubated with primary antibodies
overnight at 4 °C followed by incubation with secondary antibodies
(1:10,000) for 1 h at room temperature.Protein bands were visualized
using an ECL kit (ECL-plus, Thermo Scientific, Pittsburgh, PA, USA).