Western blot analysis

Total protein was isolated from cells or tissues with lysis buffer (APPLYGEN, Beijing, China). Protein was prepared as described previously. And 30–70 μg of protein per sample was separated via gel electrophoresis, transferred to a poly-vinylidene fluoride membrane, and blocked with 5% milk. The membrane was probed with primary antibodies overnight at 4°C, then incubated with secondary HRP-conjugated antibody at RT and exposed to enhanced chemiluminescent substrate.