P38 p p38

Protein samples were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a polyvinylidene fluoride membrane (Millipore, Darmstadt, Germany). The membrane was washed three times and blocked by soaking in Tris-buffered saline + Tween (TBS-T) buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, and 0.1% Tween 20) containing 5% bovine serum albumin (GenDEPOT) for 30 min. The membrane was incubated with primary antibodies (1:1000) overnight at 4 °C. Then, the membrane was washed three times and incubated with the appropriate secondary antibody (1:2000) for 1 h at room temperature. All primary and secondary antibodies (β-actin, Nrf2, HO-1, SOD2, catalase, Gpx-1, Bax, Bcl/Bcl-xL, JNK/p-JNK, ERK/p-ERK, and p38/p-p38) were obtained from Abcam (Cambridge, UK) and Cell Signaling Technology (Beverly, MA, USA). Protein expression was visualized using enhanced chemiluminescence reagent (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions. Quantitative analysis was conducted using ImageJ software (version 1.52a for Windows; NIH, Bethesda, MD, USA).

Fibroblasts (murine cell line, NIH-3T3 cells) obtained from the Cell Resource Center of the Shanghai Institute for Biological Sciences (Shanghai, China) were used in all experiments. Cells were cultured in DMEM and newborn bovine serum (NBS) (Invitrogen, Carlsbad, CA, United States). Other reagents (DMSO, MTT, penicillin, streptomycin, Triton X-100) were obtained commercially (Sigma-Aldrich, St. Louis, MO, United States). Mouse SA-β-galactosidase, p16, and p21 ELISA Kits were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Mouse monoclonal antibodies against p16, p21, CoL-I, PCNA, cyclin D1, cyclin E, CDK4, FAK/p-FAK, Pax/p-Pax, MEK/p-MEK, ERK/p-ERK, p38/p-p38, TGF-β1, Smad 2/3/p-Smad 2/3, GAPDH, and β-actin were purchased from Abcam. For western blotting, luminol reagent was purchased from Santa Cruz Biotechnology (Dallas, TX, United States). Sheep anti-rat secondary antibody and sheep anti-rabbit secondary antibody were purchased from Beijing Biosynthesis Biotechnology Co., LTD. (Beijing, China). All other reagents were of analytical grade and were produced in China.

The mRNA expression was analyzed by real-time quantitative polymerase chain reaction (RT-qPCR) and calculated by Livak 2^-ΔΔCt method (19 (link)). Beta-actin was used as the house-keeping gene. All primers were designed by Beacon Designer 7.0 (PREMIER Biosoft International, San Francisco, USA) and synthesized by TsingKe Biological Technology Co. Ltd. (Changsha, China). Protein expressions were analyzed by western blotting. Total proteins extracted from liver tissue lysates were separated by 10% SDS-PAGE and transferred onto polyvinylidene difluoride (PVDF) membranes. Then, PVDF membranes were blocked with skimmed milk (5% in PBS containing 0.2% Tween-20) at room temperature for 2 h and probed with primary antibodies overnight at 4°C. Antibodies for Caspase 3/Cleaved-Caspase 3, ERK/p-ERK, JNK/p-JNK, and p38/p-p38 were purchased from Abcam, Inc. (Cambridge, United States). Membranes were washed and labeled with secondary antibodies of goat anti-mouse IgG1 (Southern Biotech, USA) for 2 h at room temperature. The protein bands were visualized using ECL Prime Western Blotting Detection Reagent (Bio-Rad, USA). The chemiluminescent intensities of protein signals were quantified using Image J v1.8.0 software (National Institutes of Health, USA).