Cytoplasmic protein extraction kit

The drug-treated cells and hepatic tissue samples were washed with PBS and lysed in RIPA lysis buffer (Beyotime, Shanghai, China). The extracts were centrifuged at 12,000 rmp for 10 min at 4°C, and the supernatants were used for western blotting. the cytoplasmic and nuclear proteins were prepared with the subcellular structure cell nucleus and cytoplasmic protein extraction kit (Boster, Hubei, China) according to the manufacturer’s instruction. Protein concentration was measured using the BCA protein assay kit (Boster, Hubei, China). Equal amounts of protein were electrophoresed using an 8–15% SDS-PAGE gel and transferred to PVDF membranes. The membranes were incubated with indicated primary antibodies at 4°C overnight. After washed in TBST, membranes were incubated with the corresponding secondary antibodies conjugated with HRP for 1 h at room temperature, and then washed. Immunoreactive bands were detected using an ECL kit (NCM Biotech, Suzhou, China).

The HepG2 cell and animal liver protein samples were extracted with enhanced RIPA lysate (Boster, Hubei, China), the cytoplasmic and nuclear proteins were prepared with the subcellular structure cell nucleus and cytoplasmic protein extraction kit (Boster, Hubei, China) according to the manufacturer’s instruction. The protein concentration of whole-cell lysates was determined using the BCA method (Boster, Hubei, China). Protein lysates (15-30 μg) were loaded on 8-12% SDS-PAGE gels, separated electrophoretically and transferred to the PVDF membrane. Subsequently, the membranes were incubated in a blocking solution at room temperature for 1 h. After blocking, membranes were separately incubated at 4°C on a rocker with primary antibodies specific to the protein of interest; these were rabbit anti-Keap1 antibody (1:1000), anti-cleaved caspase3 antibody (1:1000), anti-Bax antibody (1:5000), anti-Histone H3 antibody (1:1000), anti-β-actin antibody (1:500-1:2000), mouse anti-Nrf2 antibody (1:800), and anti-Bcl2 antibody (1:500). Subsequently, the membranes were incubated with a suitable HRP-conjugated secondary antibody (Proteintech, USA) for 1h, and then signal detection was conducted with an ECL kit (Boster, Hubei, China) according to the manufacturer’s protocol.