Bcl2 associated x (bax)

GLA, diethylnitrosamine (DEN), Eagle balanced salt solution (EBSS), ponceau S, and RNase were procured from Sigma Aldrich (St Louis, MO, USA). Collagenase type 4, RNase, sodium cacodylate, hematoxylin, and eosin were purchased from Himedia. Bax and Bcl-2 were purchased from Biosynthesis Biotechnology (Beijing, China). The kits for aspartate aminotransferase (AST), alanine aminotransferase (ALT), alpha-fetoprotein (AFP), and alkaline phosphatase (ALP) were purchased from Beihuakangtai Biotechnology (Beijing, China). All other chemical and reagents used in the experimental study were acquired from the reputed vendor.

Cells were lysed using RIPA lysis buffer (Beyotime Institute of Biotechnology, China). The protein concentrations were determined using the bicinchoninic acid assay (BCA). A total of 40 μg of proteins was electrophoresed via sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, USA), which were then blocked with 5% non-fat dry milk for 1 h at room temperature. The membranes were incubated with antibodies against AEG-1 (1:1000 dilution; Abcam, USA), Bcl-2 (1: 100 dilution), Bax (1: 100 dilution), caspase-3 (1: 100 dilution; Biosynthesis Biotechnology, China), PCNA (1: 500 dilution), Cyclin D1 (1: 200 dilution), Cyclin E (1: 200 dilution), PARP (1: 500 dilution; Santa Cruz Biotechnology, USA), followed by incubation with corresponding horseradish peroxidase (HRP)-conjugated secondary antibody (Beyotime Institute of Biotechnology) at a 1:5000 dilution for 1 h at 37°C. Chemiluminescence was then produced by chemiluminescence substrate (Thermo Scientific Pierce) and subsequently exposed to X-film and scanned into the computer. Protein expression levels from each group were determined via gray scale analysis using Image J software. The β-actin served as an internal control.