Western Blot

VSMCs were lysed in lysis buffer and incubated for 30 min on the ice, the supernatant was retained after centrifugation. The total protein concentration in the supernatant was quantified with the Bradford assay (Santa Cruz, CA, USA). Equal amounts of protein in each sample were separated by SDS-PAGE electrophoresis and transferred to PVDF membrane. The membrane was blocked with 5% milk blocking buffer and incubated with the designed primary antibodies overnight at 4 °C. Horseradish peroxidase-conjugated secondary antibodies were used for detection. The protein bands were visualized with Enhanced Chemiluminescence Detection Kit (Thermo Scientific, Rockford, IL, USA).