Recombinant tgf β1

Fetal bovine serum was purchased from Biological Industries, and Dulbecco’s modified Eagle’s medium (DMEM) was purchased from HyClone. Lipofectamine^TM3,000 and Trizol were purchased from Invitrogen. β-actin, RNF2, α-SMA, IL-6, COL 1, and TNF-α primers were purchased from Invitrogen. Recombinant TGF-β1 was purchased from Novoprotein Scientific (Shanghai). RNF2 (Cat:16031-1-AP) polyclonal antibody, β-actin monoclonal antibody, HRP-conjugated goat anti-rabbit IgG (SA00001-2), and goat anti-mouse IgG (SA00001-2) were purchased from Proteintech (China). pEGFP-C2-RNF2 plasmid was stored in our laboratory. PrimeScriptTM IV 1st Strand cDNA Synthesis Mix was bought from TaKaRa. FITC Annexin V apoptosis detection kit I (401007) was purchased from BestBio. Human IL-6 ELISA KIT (ml027379), human TNF-α ELISA KIT (ml077385) and human IL-1β ELISA KIT (ml058059) were purchased from Miblo (Shanghai China). TB Green^® Premix Ex Taq^TM II was purchased from TaKaRa. COL 1 (WL0088), α-SMA (WL02510) antibodies were purchased from Wanleibio (China). ECL chemiluminescent kit was purchased from NCM Biotech. BeyoClick^TM EdU Cell Proliferation Kit was purchased from Beyotime (Cat:C0075S).

Andro was purchased from Chengdu Herbpurify Co., Ltd (Chengdu, China). Recombinant TGF‐β1 was purchased from Novoprotein (SinoBio, Shanghai, China). N‐Acetyl‐l‐cysteine (NAC) was purchased from Sigma‐Aldrich (St Louis, MO, USA). SCH772984, PD98059 and SB431542 were purchased from Selleck (Houston, TX, USA). The TRIzol reagent was purchased from Invitrogen (Carlsbad, CA, USA). The PrimeScript RT reagent Kit with gDNA Eraser was obtained from Takara Bio Inc (Shiga, Japan), and the SsoFast EvaGreen Supermix was obtained from Bio‐Rad Laboratories Inc. (Hercules, CA, USA). Antibodies to sirtuin1 (Sirt1), E‐cadherin, p‐Smad2, Smad2, p‐Smad3, Smad3, p‐Erk1/2, Erk1/2, N‐cadherin, snail, slug and vimentin were from Cell Signaling Technology (Beverly, MA, USA). Antibodies to fibronectin, matrix metalloproteinase (MMP)‐2, MMP‐9 and TGF‐β1 were from Proteintech (Chicago, IL, USA). Antibodies to α‐SMA were from Sigma‐Aldrich. Antibodies to NADPH oxidase 4 (NOX4), superoxide dismutase (SOD) 2, Forkhead box O3 (FOXO3) and GAPDH were purchased from ABclonal (Wuhan, China); all the secondary antibodies were from Abcam Biotechnology (Cambridge, MA, USA). All other chemicals and reagents used in the experiment were of analytical grade. Other reagents were all from Beyotime Institute of Biotechnology unless otherwise indicated.