Protein Extraction and Western Blot Analysis

Total protein and nuclear protein were extracted from cells and murine tissues. The protein concentration was detected by using a BCA protein assay kit. Equal amounts of protein (25 μg) were separated on SDS-PAGE gel and electro-transferred onto a polyvinylidene difluoride membrane (PVDF). Next, PVDF membranes were blocked with 5% fat-free milk for 1 h, and then incubated with primary antibodies overnight at 4 °C. After incubating with secondary antibodies at room temperature, the optical density of the bands was visualized by an ECL system (Pierce). Data was normalized to GAPDH or Histone H1 levels.