P smad2

Resveratrol was purchased from Shanghai Standardization for the Traditional Research Center and dissolved in dimethyl sulfoxide (DMSO). Human TGF-beta1 was obtained from PeproTech (USA). 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) was purchased from Promega (Madison, WI, USA). QuicBlock^TM Blocking Buffer for Immunol Staining, Antifade Mounting Medium with DAPI, QuicBlock^TM Secondary Antibody Dilution Buffer for Immunofluorescence and QuicBlock^TM Primary Antibody Dilution Buffer for Immunol Staining were purchased from Beyotime (Shanghai, China). Antibodies against MMP-2, MMP-9, α-SMA, Smad2, Smad3, P-Smad2, P-Smad3, Snail1, and Slug were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Fibronectin, P-PI3K, PI3K, P-AKT, AKT, E-cadherin, and vimentin antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). LY290042, SB431542, SP600125, PDTC, and PD98059 were purchased from Selleck Chemical (Houston, TX, USA). PDTC and PD98059 were purchased from MedChemExpresss (New Jersey, NJ, USA). IRDye^TM fluorescence antibodies were obtained from Li-Cor Bioscience (Lincoln, NE, USA).

The concentration of protein in the lysed sample was analyzed using a bicinchoninic acid assay kit (Thermo Scientific, Pierce, Rockford, IL) after heating at 95°C for 5 min. First, the desired volume of samples was loaded in 10% SDS-polyacrylamide gels and run at a constant voltage. The separated proteins were then transferred to a nitrocellulose membrane (Trans-Blot Turbo^TM, BIO-RAD). Subsequently, the transferred membrane was cut at the desired part and incubated with a blocking buffer before incubation with the desired antibodies.
The level of p-SMAD2, SMAD7, ATG5, p62, p-AKT, p-mTOR, Bcl2, Bax, caspase-3, β-actin (Santa Cruz Biotechnology (Dallas, TX, USA), LC3BI/II, p-p38, p-ERK, α-SMA, Col1A1, and p-IκBα (Cell Signaling Technology, Beverly, MA, USA) were evaluated by incubating (4°C) the membrane overnight with the appropriate primary antibodies at a 1:1000 dilution of their original antibodies. The goat anti-rabbit and anti-mouse IgG-HRP polyclonal antibodies (AbFrontier, Seoul, South Korea) were used as the secondary antibodies. The optical protein bands were detected by adding a mixture (1:1 ratio) of a western blot detection solution A and B (SUPEX, Neonex Co., Ltd, Postech, South Korea), after which the area of the densitogram peak was estimated using the Image J software (National Institute of Health, Bethesda, MD, USA).

LPS (Sigma; Escherichia coli serotype 055:B5), recombinant human TGF-β1 (R&D Systems) were used in this study. The antibodies used for the western blot analysis, and are p-JNK (catalog no. sc-81502), JNK (catalog no. sc-7345), p38 (catalog no. sc-398305), p-p38 (catalog no. sc-17852-r), p-Smad2 (catalog no. sc-101801), Smad2 (catalog no. sc-39312), p-Smad3 (catalog no. sc-101154), and Smad3 (catalog no. sc-130218), all purchased from Santa Cruz Biotechnology, Inc. NF-κB inducible reporter plasmid were purchased from InvivoGen (cat no: pnifty2-luc, San Diogo, CA). Lipofectamine 2000 transfection regent was purchased from Invitrogen.