β actin

The western blot analysis was done according to a published method [33 ]. Proteins were extracted from liver homogenates by SDS-polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane (Millipore, Bedford, MA) for 1 h at room temperature. The membranes were blocked with TBST containing 10% skimmed milk for 2 h at room temperature and then probed with the primary antibody against NFκB p65 (1:1000) (Santa Cruz Biotechnology, California, US), IκB (1:1000) (Santa Cruz Biotechnology, California, US), β-actin (1:5000) (Easybio, Beijing, China), and Histone H3 (1:5000) (Easybio, Beijing, China) overnight at 4 °C. After three 10-minute washes in TBST, the membranes were incubated with the horseradish peroxidase-conjugated secondary antibody (1:10000) at room temperature for 60 min. The membranes were washed with TBST three times. The protein bands were then visualized with an enhanced chemiluminescence detection kit (Beyotime, Shanghai, China). The blots were analyzed by scanning densitometry with a GS-700 imaging densitometer and Quantity One software. The optical density ratio of the target proteins to their internal reference proteins was used as the relative expression of the target protein.