Anti smad2 3

Whole cell protein extracts were obtained by direct lysis in Lammelli’s Buffer and boiled at 100°C for 10 min. Proteins were separated on 12% SDS-polyacrylamide gels and transferred to PVDF membranes (#10600023, Amersham Biosciences, UK). Nonspecific binding sites were blocked for 1 hour at room temperature with 5% nonfat dry milk in TBS-T. Membranes were incubated overnight at 4°C with anti-SMAD2/3 (1:1000, #8685, Cell Signaling Technology, Germany) and anti-GAPDH (1:2000, #2118, Cell Signaling Technology). Membranes were washed 3 times with 1% TBS-T, followed by incubation with HRP-conjugated anti-rabbit secondary antibodies (1:1000, #7074, Cell Signaling Technology). Protein complexes were visualized with enhanced chemiluminescent HRP substrate (#R-03031-D25 and R-03025-D25, advansta, USA) and bands were quantified using ImageJ software (National Institute of Mental Health).

Protein lysates from heart samples were loaded on and separated by a 10–15% SDS-PAGE, and then transferred to PVDF membranes (Millipore, MA, United States). The membranes were probed with primary antibodies overnight at 4°C after blocking with 5% milk in Tris-buffered saline with 0.1% Tween (TBST). The primary antibodies were anti-TGFβ1, anti-TGFβ receptor I (TGFβRI), anti TGFβ receptor II (TGFβRII), anti-phosphorylated Smad2/3, anti-Smad2/3, and anti-Smad4 (Cell Signaling Technology, shanghai, China), anti-collagen I and III (Col I and III, Proteintech, Wuhan, China), anti-α-SMA, anti-periostin, anti-ACE (Abcam, Shanghai, China) and anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Multi Sciences, Hangzhou, China). Membranes were washed by TBST for three times and then incubated with appropriate secondary antibodies for 1 h at room temperature. After another three times wash by TBST, the signal was detected on FluorChemE (Protein Simple, CA, United States). The densities of bands were quantified by an ImageJ Analysis System and expressed as ratios to GAPDH.

Dulbecco's modified Eagle's medium (DMEM), fetal calf serum, and tissue culture reagents were obtained from Invitrogen/GIBCO (Grand Island, NY, USA). Momordicine I (>99% purity, kindly provided by Dr. Shi-Yie Cheng, Department of Life Sciences, College of Science, National University of Kaohsiung, Kaohsiung, Taiwan, ROC) was dissolved in dimethyl sulfoxide (DMSO), and the DMSO content in all groups was 0.1%. Anti-p-Smad2/3, anti-Smad2/3, anti-GAPDH, and anti-PARP antibodies were purchased from Cell Signaling Technology (Boston, MA, USA). Anti-HO-1 and anti-Nrf2 antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Brusatol, the Nrf2-specific inhibitor, and all other reagent-grade chemicals were acquired from Sigma-Aldrich Chemical Co. (St. Louis, MO, USA).

Human HSC LX2 was received as a kind gift from Prof. Scott Friedman. Human fetal hepatocytes LO2 was received as a kind gift from A/Prof. Victor Yu. MgIG was obtained from Jiangsu Chia-Tai Tianqing Pharmaceutical Co., Ltd. (Nanjing, China). Compound structure and detailed information is supplied in Supplementary Figure S1. PCR primers were purchased from Integrated DNA Technologies (Coralville, IA, United States). The following antibodies were used: Anti-collagen-1 was purchased from Abcam (Cambridge, United Kingdom), anti-αSMA from Agilent Dako (Santa Clara, CA, United States); anti-GAPDH, anti-phospho-ERK, anti-ERK, anti-phospho-Akt, anti-Akt, anti-phospho-JNK, anti-JNK, anti-SMAD2/3, anti-SMAD4, secondary anti-mouse and anti-rabbit were purchased from Cell Signaling Technology (Danvers, MA, United States); anti-phospho-p38, anti-p38 and anti-p27 were purchased from Santa Cruz Biotechnology (Dallas, TX, United States). 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) was purchased from Duchefa Biochemie (Haarlem, Netherlands) and propidium iodide (PI) was purchased from Sigma-Aldrich (St. Louis, MO, United States). 96-well cellular senescence assay kit was purchased from Cell Biolabs (San Diego, CA, United States).