Western Blotting

The total proteins of hepatocytes were extracted by radio immunoprecipitation assay (RIPA) lysate (ThermoFisher, Waltham, USA) containing both protease inhibitor and phosphatase inhibitor (Beyotime, Shanghai, China). Briefly, the concentrations of protein samples were detected by using Protein Assay Kit (Beyotime, Shanghai, China). A volume of 30 μg proteins from each sample was separated by 10% to 15% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF). The films were soaked with phosphate buffer saline tween (PBST), blocked with 5% skimmed milk powder solution at 4°C for overnight, then incubated with the primary antibodies of TNF-α (26 kDa; Mouse Anti-TNF alpha monoclonal antibody; 1:1000; bsm-33207M; Bioss, Beijing, China), p-JNK (50kDa; Rabbit Anti-phospho-JNK1 (Thr183) polyclonal antibody; 1:1000; bs-17591R; Bioss, Beijing, China), JNK (42 kDa; Rabbit Anti-JNK1+JNK3 polyclonal antibody; 1:1000; bs-20760R; Bioss, Beijing, China), Nrf2 (110kDa; Rabbit Anti-Nrf2 polyclonal antibody; 1:1000; bs-1074R; Bioss, Beijing, China) or glyceraldehyde-3-phosphate dehydrogenase (GAPDH, 36 kDa; mouse anti-GAPDH-loading control antibody; 1:5000; ab8226; Bioss, Beijing, China) gently shaken for 2 h at room temperature. The films were further incubated with the HRP-conjugated goat anti-rabbit antibody (1:5000; ab205718; Beyotime, Shanghai, China) or HRP-conjugated goat anti-mouse antibody (1:5000; ab205719; Beyotime, Shanghai, China) for 2 h at room temperature. Finally, SignalFire plus ECL reagent (ThermoFisher, Waltham, USA) was used to show the target bands, then the ImageJ software (version 5.0, BIO-RAD, California, USA) was used to analyze and count the gray value of the bands. The target band was normalized to GAPDH in each lane, and each plate was homogenized by the first hole.