Western blot analysis

Total protein or nuclear protein was extracted using the EpiQuik whole-cell extraction kit or EpiQuik nuclear protein extraction kit (Epigentek, USA). The protein concentration was measured following the manufacturer's instruction (Bio-Rad, USA). Protein was applied and separated on 4–15% NuPAGE gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes (Millipore, USA). The membranes were blocked with 5% bovine serum albumin and incubated with specific antibodies (1:1,000 dilution) overnight. Horseradish peroxidase-conjugated IgG (1:10,000 dilution) from Santa Cruz Biotechnology (Santa Cruz, USA) was used to treat the membrane for 1 h, after which the membranes were enhanced with a SuperSignal West Pico Chemiluminescent Substrate (Thermo, USA). The relative amounts of the transferred proteins were quantified with scanning the autoradiographic films. Total protein or nuclear protein was normalized to the corresponding β-actin or PCNA level.