Liver cells were lysed by RIPA (Beyotime, Guangzhou, China) and centrifuged at 12,000 × g for 5 min. The supernatant which contains the protein was collected. The protein in hepatocytes nucleus was extracted with the commercial Kit manufactured by Beyotime Biotechnology. The protein concentrations were determined with the BCA Assay Kit from Beyotime Biotechnology. CYP2A5, Nrf2 (mouse and human), and CYP2A6 proteins were separated by SDS-polyacrylamide gel electrophoresis (12%), electrophoretically transferred to nitrocellulose/polyvinylidene difluoride membranes (Pierce Biotechnology, Rockford, IL/Bio-Rad Laboratories, Hercules, CA), and blocked in phosphate buffer containing 0.1% Tween 20 and 5% non-fat milk for 1 h at RT. Blots were incubated with Nrf2 polyclonal antibody (rabbit anti mouse, 1:500 diluted, Bioss, Beijing, China), CYP2A5 monoclonal antibody (chicken anti mouse, 1:1000 diluted, presented by University of Oulu), CYP2A6 polyclonal antibody (rabbit anti human, 1:200 diluted, ImmunoWay Biotechnology Company), and Nrf2 polyclonal antibody (rabbit anti human, 1:200 diluted, ImmunoWay Biotechnology Company) for 1 h at 37°C. Membranes were then incubated with horseradish peroxidase-conjugated antibody (goat anti rabbit, 1:5000 diluted, Boster biological engineering co., LTD, Wuhan, China; rabbit anti chicken, 1:5000
