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.