Gapdh

Liver tissues (30–100 mg) were homogenized and the protein liquid was extracted. The proteins were separated and transferred to the polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The PVDF membranes were incubated with primary antibody overnight at 4°C. The antibodies included: α-SMA (1:1,000; Servicebio Technology, Wuhan, China), fibronectin (FN), collagen I and connective tissue growth factor (CTGF) (1:1,000; BOSTER Biological Technology, Wuhan, China), TGF-β1 and Smad2/3 and P-Smad2/3 (1:1,000; Cell Signaling Technology Inc., Danvers, MA), GAPDH and β-actin (1:1,000; Aspen Biological, Wuhan, China). Then, the PVDF membranes were incubated with the anti-rabbit IgG secondary antibody (1:6,000; Aspen Biological, Wuhan, China) at room temperature for 1 hour. Finally, the proteins were detected with the Super Signal West Pico plus Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA).

Total protein was extracted after cell lysis by RIPA lysis buffer (Beyotime Biotechnology, Shanghai, China), and the protein concentration was detected using a BCA protein assay kit (Beyotime). Equal amounts of protein were added to the SDS polyacrylamide gel and transferred to a PVDF membrane (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk powder in TBST solution for 1 h and then incubated with primary antibodies overnight at 4 °C. Antibodies for the following were used and diluted following the manufacturer’s instructions: Bmi1 (#6964, 1:1000), AKT (#9272, 1:1000), p-AKT (phospho Ser473,#4060, 1:2000), p38 MAPK (#8690, 1:1000), p-p38 MAPK (phospho Thr180/Tyr182, #4511, 1:1000), STAT3 (#4904, 1:2000), and P-STAT3 (phospho Y705; #9145, 1:2000). The antibodies were purchased from Cell Signaling, Danvers, MA, USA, and GAPDH (AS1039, 1:1000) was purchased from Aspen, Wuhan, China. After washing three times with TBST for 10 min each time, the membranes were incubated with secondary horseradish peroxidase-coupled antibody (Aspen), visualized using an ECL kit (ThermoFisher, Waltham, MA, USA) and exposed to a gel imager. GAPDH was used as an internal control, and the gray value of each protein was calculated.

The CHON-001 cells were seeded in 96-well plates. The cells were then lysed using radioimmunoprecipitation assay buffer for 30 min on ice. Proteins were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride membranes. The membranes were blocked with 5% skimmed milk for 2 h and then cultivated with primary antibodies against CCR10, cleaved-caspase-3, MMP-3. MMP-13, Collagen II, Aggrecan, p-PI3K, PI3K, p-Akt, Akt, p-mTOR, mTOR, GAPDH, or β-actin (1:1000, ASPEN) at 4 °C overnight. After washing thrice with Tris-buffered saline with 0.1% Tween® 20 detergent, the membranes were incubated with secondary antibodies for 2 h. Protein signals were visualized using enhanced chemiluminescence detection reagents and quantified using ImageJ software.