2.9 Immunoprecipitation and western blot analysis
RIPA lysate buffer (Beyotime Biotechnology, P0013B) containing 1% PMSF was used to extract the total proteins from cells and hepatic tissues. Protein concentrations were measured using a BCA kit (Beyotime Biotechnology, P0009). After immunoprecipitating, proteins with the appropriate antibodies overnight at 4°C and incubating the complexes with Protein A/G Plus‐agarose (Santa Cruz Biotechnology, sc‐2003) for 4 hr at 4°C, total cell lysates were washed five times, mixed with 2× SDS loading buffer, and boiled for 10 min. Equal amounts of immunocomplexes or lysates were electrophoresed on SDS‐PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% skim milk in PBS‐T buffer (PBS containing 0.2% Tween 20) at room temperature for 1 hr and then immunoblotted with primary antibodies overnight at 4°C. Peroxidase‐conjugated secondary antibodies were then applied to the membranes and incubated for 1 hr at room temperature. Electrochemiluminescence was performed according to the manufacturer’s instructions with a BIO-RED ChemiDoc XRS+ imaging system. β-actin was served as an internal control. Both immunoprecipitation assay and western blot have been conducted the experimental detail provided conforms with BJP Guidelines (Alexander et al., 2018). The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology.