Cleaved Caspase-3 Levels in Rat Liver

Protein levels of cleaved caspase-3 in rat livers were determined by Western blot analysis. Proteins extracted from livers were subjected to SDS-PAGE gels and electrotransferred to polyvinylidene fluoride (PVDF) membranes. Nonspecific binding sites were blocked in 5% bovine serum albumin at room temperature for 1 h. Membranes were subsequently probed with primary antibodies at 4 °C overnight, then incubated with the appropriate peroxidase-conjugated secondary antibody. Cleaved caspase-3 monoclonal antibody (1:1000; Cell Signaling Technology, Boston, MA, USA) and Cyp7a1 antibody (1:1000; Shenggong BBI Life, Shanghai, China) were used as primary antibodies, and horseradish-peroxidase-conjugated anti-rabbit IgG (1:5000; Abclonal, Wuhan, China) was used as a secondary antibody. Densitometry of immunoblot analysis was performed using ImageJ software as previously described [14 (link)].