Smad7

Cells were lysed by radioimmunoprecipitation buffer, and then cell lysates were separated by SDS‐PAGE. After blocking, the membrane was incubated with primary antibodies (Smad7 and GAPDH, Abcam) and the secondary antibodies (Rockland, Limerick, PA, USA). Protein levels were normalized to that of total GAPDH.

TAA and silymarin were obtained from Sigma Aldrich (St. Louis, Missouri, USA). The ELISA kits used to measure alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and r-glutamyl transferase (r-GPT) were acquired from Abcam (Cambridge, UK). Malondialdehyde (MDA), glutathione (GSH), catalase (CAT), and superoxide dismutase (SOD) assay kits were purchased from Cayman Chemical (Ann Arbor, Michigan, USA). Antibodies against collagen-1, TGF-β1, α-SMA, vimentin, Smad2/3, p-Smad2/3, Smad4, Smad7, p-PTEN, PTEN, p-Akt, Akt, p-PI3K, PI3K, and β-actin were purchased from Abcam (Cambridge, Massachusetts, USA) and Cell Signaling Technology (Danvers, Massachusetts, USA). Anti-mouse and anti-rabbit IgG and HRP-linked-conjugated secondary antibodies were procured from Santa Cruz Biotech. (Santa Cruz, California, USA).

RNF12 and related expression constructs were cloned and verified via DNA sequencing. RNF12 S215A and RNF12 S347A/T349A were generated by site-directed mutagenesis and confirmed via DNA sequencing. Myr-AKT1 constructs were kindly provided by P. Coffer (University Medical Center, Utrecht, The Netherlands). The reagents used were IGF-1 (R&D 291-G1), MK2206 (Selleck, S1078), MG132 (Selleck, S2619), cycloheximide (CHX) (Sigma, C104450), and SB431542 (Millipore, 616461). The antibodies used for immunoprecipitation (IP), immunoblotting (IB) and immunofluorescence were, c-Myc (IB; no.9402, Cell Signaling), HA (IB; Y-11, sc-805, Santa Cruz), HA (IB; 12CA5, home-made), Flag (IB; M2, Sigma), β-actin (IB; A5441, Sigma), RNF12 (IB, CST) (IP; U0635, Proteintech), AKT (IB, Cell Signaling) (IP; no. 2938, Cell Signaling), phosphor-AKT substrate (RXRXXS*-T*) (IB; no. 10001, Cell Signaling), phosphor-AKT (Ser473) (IB; no. 9271, Cell Signaling), tubulin (IB; no. 2146, Cell Signaling), SMAD2-3(IB; 610842 BD), phospho-SMAD2/3(IB; no. 3101, Cell Signaling), SMAD7(IB; ab216428, Abcam)(IB; MAB2009, R&D), Ub (IB; P4D1, Santa Cruz), SNAIL (IB; C15D3, Cell Signaling), and SLUG(IB; C19G7, Cell Signaling). RNF12 Ser215 phosphorylation-specific antibody was produced in a biotechnology company (GL Biochem Ltd, Shanghai).

Tissues were lysed with ice-cold RIPA lysis buffer containing 1% PMSF after centrifugation as described previously. Protein concentrations in the samples were measured with a spectrophotometer. Antibodies used in this study included GAPDH and Smad7 (Abcam, Cambridge, MA, USA). The total protein (20 μg) was separated by 10% SDS polyacrylamide gel electrophoresis. The resolved proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane, which was subsequently blocked for 3 h at room temperature with 5% skim milk in TBS-T (10 mM Tris, 150 mM NaCl, and 0.1% Tween-20). After washing with TBS-T, the membrane was incubated with the following antibodies overnight at 4°C: anti-Smad7 at a 1 : 1000 dilution and anti-GAPDH at a 1 : 10000 dilution. The secondary antibodies (Jackson, USA) (goat anti-rabbit and goat anti-mouse IgG horseradish peroxidase conjugated antibody) were used at a 1 : 4000 dilution to detect the target protein. The reaction was visualized on X-ray medical film after incubation of the membranes with luminol-based chemiluminescence reagent (Invitrogen, USA) for 5 min. Experiments were carried out in triplicate at least three times.

Cultured HDFs were lysed with radio-immunoprecipitation assay lysis buffer (Beyotime, Jiangsu, China). Protein concentrations were determined using a micro bicinchoninic acid assay (Thermo Fisher Scientific). Total proteins were separated on acrylamide gels, and immunodetected with the primary antibodies that included the following: Col1a2, Col3a1 (Genetex, Biozol, Eching, Germany, 1:1000), α-SMA, Smad6, Smad7, TGF-β receptor I (TGFβRI), TGF-β receptor II (TGFβRII) (Abcam, Cambridge, UK, 1:1000), Smad2, p-Smad2, Smad3, p-Smad3, Smad4, Smad1/5/9, p-Smad1/5/9 (Cell Signaling Technology, Beverly, MA, 1:1000). Immunoreactive bands were quantitatively analyzed with ImageJ software.

The Western blotting protocol was described in our previous study. Briefly, myocardial tissue was lysed in ice-cold RIPA buffer (150 of mM sodium chloride, 0.1% sodium dodecyl sulphate (SDS), 0.5% sodium deoxycholate, 1.0% NP-40, PMSF 1 mM, and 50 mM of Tris, pH 8.0) for total protein extraction. The total protein concentration was quantified by a BCA Protein Assay Kit (Medchem Express, USA). Equal amounts of protein were separated by 12% SDS–polyacrylamide gel electrophoresis (PAGE). Then, the protein was transferred from the gel to a polyvinylidene fluoride membrane. After blocking with 5% skim milk, the membrane was incubated overnight in primary antibody (Nrf2, HO-1, TGF-β1, p-Smad2, Smad2, p-Smad3, Smad3, Smad7, α-SMA 1:1000, Abcam). Bands were detected with specific horseradish peroxidase-conjugated secondary antibody (CWBIO). β-actin (1:1000, Abcam) was used as a reference of total cell protein. The density of the signal was quantified by ImageJ (version 1.8.0).

Total protein of left ventricular tissue was extracted with RIPA lysis buffer (Beyotime Biotech, Haimen, China) supplemented with 1 mmol/L PMSF, followed by determination of protein concentrations with BCA protein assay kit. Then protein samples were subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and transferred to nitrocellulose membrane (Millipore, MA, USA). After blocking with 5% skimmed milk in Tris buffered saline containing 0.5% Tween-20 (TBST), the membrane was incubated overnight at 4°C with primary antibodies against superoxide dismutase (SOD, 1:1000), Smad3 (1:3000), p-Smad3 (phospho Ser 423/425, 1:2000), Smad7 (1:1000, Abcam, Cambridge, UK) and tubulin (1:1000, Beyotime Biotech, Haimen, China), followed by incubation with horseradish peroxidase (HRP) conjugated secondary antibody (Beyotime Biotech, Haimen, China) for 1 h at room temperature. Bound HRP was visualized on an X-ray film with an enhanced chemiluminescence substrate (ECL, Beyotime Biotech, Haimen, China). Protein bands were analyzed with Quantity One software (Bio-Rad, USA) and the level of each protein was normalized to that of tubulin except for that of p-Smad3, which was normalized to that of Smad3.