Collected liver tissue was homogenized on ice with RIPA (P0013B, Beyotime technology, Shanghai, China) containing 10% protease inhibitor cocktail (Roche, Mannheim, Germany), phosphatase inhibitor cocktail III (C0004, TargetMol, Boston, MA, USA) to extract the total proteins. A Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to quantify the concentrations of the proteins according to the standard instructions. Each protein sample (15 μg) underwent 12% SDS-PAGE separation and was transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Then, the blots were blocked with 5% skim milk for 2 h at room temperature and incubated 10–12 h at 4 °C with the following primary antibodies shown in Table 2 .
Then, the membranes were incubated with goat anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution, Santa Cruz Biotechnology) at room temperature for 2 h. The signals were visualized with ECL detection reagents (Millipore, Billerica, MA, USA). The relative expression of the protein was determined by using a ChemiDoc™ XRS+ System and quantified by Image Lab v.4.0 software (Bio-Rad, Hercules, CA, USA).
Then, the membranes were incubated with goat anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibodies (1:5000 dilution, Santa Cruz Biotechnology) at room temperature for 2 h. The signals were visualized with ECL detection reagents (Millipore, Billerica, MA, USA). The relative expression of the protein was determined by using a ChemiDoc™ XRS+ System and quantified by Image Lab v.4.0 software (Bio-Rad, Hercules, CA, USA).