T jnk

LicA (BP0855) was purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China). Primary antibodies against p-ERK, cleaved caspase-3, cleaved caspase-9, and cleaved poly (ADP-ribose) polymerase (PARP) were bought from Cell Signaling Technologies (Beverly, MA, USA). Primary antibodies against Bcl-2, Mcl-1, Bax, t-ERK, p-p38, t-p38, p-JNK, t-JNK, β-actin, and siRNA-p38 (sip38) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Moreover, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Z-VAD-FMK and tauroursodeoxycholic acid (TUDCA) were purchased from BioVision (Milpitas, CA, USA). BIRB 796 was bought from Tocris Bioscience (Minneapolis, MN, USA). Fetal bovine serum (FBS) was purchased from HyClone (Logan, UT, USA).

Chemical inhibitors were purchased from Calbiochem (LY294002, PD98059, SB203580), Sigma (SP600125), and SelleckBiochem (A66, TGX-221, CAL-101, AS-252424, Ruxolitinib). The doses used and specific targets for each inhibitor are summarized in Table 1. Recombinant proteins were purchased from Austral Biological (IGF-1) and R&D Systems (IL-1β, OSM, IL-6, sIL-6R). Antibodies were purchased from Cell Signaling Technology (p-Akt S473, T-Akt, p-ERK T202/Y204, T-ERK, p-JNK T183/Y185, T-JNK, p-p38 T180/Y182, T-p38), Santa Cruz Biotechnology (p-STAT3 Y705, T-STAT3), and Abcam (MMP-13, MMP-2).

Total cell lysates were prepared with protein lysis buffer (Intron Biotechnology, Seoul, Korea) supplemented with 1× protease inhibitor cocktails and PMSF (Sigma-Aldrich, St. Louis, MO, USA). The lysates were suspended with sample buffer containing sodium dodecyl sulfate (SDS) and boiled for 7 min for denaturation. Proteins were separated on 8% or 12% SDS-polyacrylamide gels electrophoresis, and then transferred onto a polyvinylidene fluoride (PVDF) membranes. The membrane was incubated with primary antibodies in Tris-buffered saline containing 0.1% Tween-20 (TBS-T) for overnight at 4 °C. Primary antibodies for p-ERK1/2, t-ERK1/2, p38, p-JNK, t-JNK, AKT, and β-actin were purchased from Santa Cruz biotechnology Inc. (Santa Cruz, CA, USA). p-p38 and p-AKT antibodies were obtained from (Cell Signaling Technology Inc., Danvers, MA, USA). The membrane washed with TBS-T to remove primary antibody, followed by incubation with specific secondary antibody (Jackson ImmunoResearch Laboratories Inc., West Grove, PA, USA) for 2 h. The signals were visualized using enhanced chemiluminescence (ECL; Abclon, Seoul, Korea) and an Image Quant LAS-4000 (Fujifilm Life science, Tokyo, Japan).

Small pieces of liver tissues were lysed and ∼80 μg of proteins were used and analyzed by immunoblot. Antibodies against c-Jun (9165; CST), Acta-2 (M0851; Dako), Desmin (14026; Santa Cruz), Vimentin (5741; CST), TGF-β1 (130348; Santa Cruz), TNF-α (52746; Santa Cruz), Gapdh (25778; Santa Cruz), p-JNK (9255; CST), and t-JNK (7345; Santa Cruz) were used in this work. Multiple gels were run with the same set of lysates from each cohort of mice to be probed with the various antibodies, with one or two blots being used for assessment of loading using the anti-Gapdh antibody.

Pra-B (purity > 98%) was purchased from ChemFace (Hubei, China). The origin stock solution of Pra-B is 100 mM in DMSO solvent. The final DMSO concentrations ranged from 0.01% for 10 μM Pra-B to 0.05% for 50 μM Pra-B. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies for CTSC, CTSV, t-ERK, p-JNK, t-JNK, p-p38, t-p38, p-MEK, t-MEK, p-EGFR, t-EGFR, and β-actin were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The antibody for p-ERK was purchased from Cell Signaling Technology (Danvers, MA, USA).