Secondary horseradish peroxidase conjugated igg antibodies

Total protein was extracted from TSCC cells using RIPA lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) and collected by centrifugation at 10,000 × g for 10 min at 4°C. SDS-PAGE (using a 12% gel) was performed with 40 µg protein/sample. The proteins were transferred to PVDF membranes that were subsequently blocked for 1 h at room temperature using 5% skimmed milk powder. The membranes were then incubated with primary antibodies against p21 (1:500 dilution; cat. no. D153319; Sangon Biotech Co., Ltd.) and β-actin (1:1,000 dilution; cat. no. sc-47778; Santa Cruz Biotechnology, Inc.) at 4°C overnight, followed by incubation with secondary horseradish peroxidase-conjugated IgG antibodies (1:5,000 dilution; cat. no. A0216 and A0208; Beyotime Institute of Biotechnology) at 37°C for 45 min. The membranes were developed using ECL Plus reagent (cat. no. P0018; Beyotime Institute of Biotechnology) and the relative expression levels were quantified using the Gel-Pro-Analyzer software version 4.0 (Media Cybernetics, Inc.) with β-actin as the internal reference.

Liver tissues or cells were directly lysed for 30 min on ice with an extraction buffer (T-PER or RIPA; Thermo Fisher Scientific Inc.). Protein concentration was determined using Pierce BCA Protein Assay kit (Thermo Fisher Scientific Inc.) according to the manufacturer's protocol. Following sodium dodecyl sulfate–polyacrylamide gel electrophoresis, gels were transferred to a polyvinylidene difluoride membrane, and then blocked with 5% bovine serum albumin in Tris-buffered saline (20mM Tris, 150mM NaCl, pH 7.4) with 0.05% Tween-20 for 1 h at room temperature. Primary antibodies were diluted at 1:1,000 in blocking solution and incubated overnight at 4°C. Antibodies specific for CYP2E1 (Abcam, Cambridge, UK), c-Jun N-terminal kinase (JNK), phospho JNK (pJNK), and β-actin (Cell Signaling Technology, Danvers, MA, USA) were used. Secondary horseradish peroxidase-conjugated IgG antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used to detect antigen–antibody complexes on polyvinylidene difluoride membranes. Immunoblot images were visualized with ImageQuant LAS 500 (GE Healthcare Life Science, Pittsburgh, PA, USA). The expression levels of protein bands were quantified with ImageQuant TL software (GE Healthcare Life Science).