2.10 Western blot analysis
Radioimmunoprecipitation assay (RIPA) lysis and extraction buffer
containing 1% Phenylmethanesulfonyl fluoride (PMSF) was utilized to
extract total protein from damaged tissue and cells. Protein
quantification was performed using the BCA kit (Beyotime, Shanghai,
China). Equal amounts of protein (30–50 µg) from each sample were
separated using an 8-12% sodium dodecyl sulfate SDS-PAGE gel and
subsequently transferred onto polyvinylidene difluoride (PVDF)
membranes. The membranes were then blocked with a 5% skim milk solution
in TBST for one hour at room temperature, followed by overnight
incubation with specific primary antibodies at 4°C on a shaker.
Subsequently, the membranes were subjected to three washes with TBST and
then probed with HRP-conjugated secondary antibody that was diluted in
TBST for 1 hour at room temperature while being constantly agitated. The
protein bands were visualized using an ECL substrate (Thermo Fisher
Scientific, Waltham, USA) and imaged under a detection system (Bio-Rad,
Hercules, California, USA). The optical densities of the bands were
scanned and quantified using image-analysis software (Image J software,
National Institutes of Health, USA), with β-actin serving as an internal
control.