Anti lamin b

We extracted the protein (including total, nuclear and cytoplasmic protein) of the cells using RIPA lysis buffer (50 mM Tris-HCl pH 8.0, 150 mM NaCl, 1%Triton X-100, and 1 protease inhibitor cocktail tablet/10 ml) and detected the protein concentration with a BCA kit (Beyotime, China). The western blotting was conducted as previously described [23 (link)]. The primary antibodies were anti-E-cadherin (Bioss, USA), anti-N-cadherin (Santa Cruz, USA), anti-Vimentin (CST, USA), anti-GSK-3-β (Bioss, USA), anti-β-Catenin (CST, USA), anti-Twist1 (Sigma, USA), anti-GAPDH (Santa Cruz, USA), and anti-Lamin B (Bioss, USA).

For nuclear factor erythroid 2-related factor 2 (Nrf2) expression analysis, the extraction and isolation of cytoplasmic and nuclear protein were performed using a Cytoplasmic and Nuclear Protein Extraction Kit (Beyotime, Nanjing, China) according to the manufacturer’s instructions. For CYP2E1 expression analysis, the extraction and isolation of microsomal protein were carried out as described previously [10 (link)]. The concentration of protein was determined by BCA assay kit (Beyotime, Nanjing, China). Equal amounts of protein extracts were subjected to SDS/polyacrylamide gel electrophoresis under reducing conditions on concentrate protein gel 5% (pH = 6.8) and separating protein gel 12% (pH = 8.8). The separated proteins were transferred to PVDF membranes using a tank transfer for 2 h at 200 mA in Tris-glycine buffer with 15% methanol. Membranes were blocked with 5% skimmed milk for 3 h and incubated for 12 h with anti-CYP2E1 (1:1500, BOSTER, Wuhan, China), anti-Nrf-2 (1:500, Bioss, Beijing, China), anti-GAPDH (1:1000, BOSTER, Wuhan, China) and anti-Lamin B (1:500, Bioss, Beijing, China) for 2 h at 37 °C. The secondary antibodies (IgG/HRP) were incubated for 2 h at 37°C. The images of the blots were visualized by ECL (Genshare, Xi’an, China).

For Nrf2 expression analysis, the extraction and isolation of cytoplasmic and nuclear proteins were performed using a Cytoplasmic and Nuclear Protein Extraction Kit (Beyotime, Nanjing, China), according to the manufacturer’s instructions. For CYP2E1 expression analysis, the extraction and isolation of microsomal proteins were carried out as described previously (Jiang et al., 2016 (link); Chen et al., 2019 (link)). The protein concentration was determined by BCA assay kit (Beyotime, Nanjing, China). Equal amounts of protein extracts were subjected to SDS–polyacrylamide gel electrophoresis under reducing conditions in concentrate protein gel 5% (pH = 6.8) and separating protein gel 12% (pH = 8.8). The separated proteins were transferred to PVDF membranes using tank transfer for 2 h at 200 mA in Tris–glycine buffer with 15% methanol. Membranes were blocked with 5% skimmed milk for 3 h and incubated for 12 h with anti-CYP2E1 (1:1500, Boster, Wuhan, China), anti-Nrf-2 (1:500, Bioss, Beijing, China), anti-GAPDH (1:1000, Boster, Wuhan, China), and anti-Lamin B (1:500, Bioss, Beijing, China) for 2 h at 37°C. The secondary antibodies (IgG/HRP) were incubated for 2 h at 37°C. The images of the blots were visualized by ECL (Genshare, Xi’an, China).