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.