2.8. Nuclear Protein Extraction and Western Blot Analysis

Nuclear extracts were prepared according to the manufacturer's manual (Sigma, St. Louis, MO, USA). Harvested cells were lysed in buffer A on ice for 10 min and then centrifuged at 17000 g for 10 min at 4°C. The nuclear pellets were resuspended in buffer B on ice for 40 min and then centrifuged at 17000 g for 10 min at 4°C. The supernatants containing nuclear extracts were stored at –80°C until use.

Protein concentration was determined by using a dye-binding protein assay kit (Beyotime, Guangzhou, China). Protein extracts were separated by 10% SDS-PAGE and transferred to PVDF membranes (Amersham Pharmacia Biotech, Little Chalfont, UK). The membranes were blocked at room temperature for 1 h with 5% nonfat dry milk and then incubated with each primary antibody at 4°C overnight and further incubated for 1 h with HRP-conjugated secondary antibody. Bound antibodies were detected by the ECL system with a Lumi Vision PRO machine.