Recombinant human tgf β2

Recombinant human TGF-β2 was purchased from Wako Pure Chemical Industries (Osaka, Japan). Y-27632 and SB203580 were purchased from Merck Millipore (Darmstadt, Germany). The anti-COL1A2 (1:2,000 dilution;), anti-Caveolin 1 (1:200 dilution), and anti-myocilin (1:100 dilution) antibodies were obtained from Abcam (Cambridge, UK). The anti-MLC2 (1:800 dilution), anti-phospho-MLC2 (1:700 dilution; Thr18/Ser19), anti-Smad2/3 (1:1,000 dilution), anti-phospho-Smad2 (1:1,000 dilution; Ser465/467), anti-α-tubulin (1:1,000 dilution), and anti-lamin A/C (1:2,000 dilution) antibodies were purchased from Cell Signaling Technology (Danvers, MA). The anti-β-actin mouse monoclonal antibody (1:10,000 dilution;) was obtained from Sigma (St. Louis, MO). The anti-collagen 4 α 5 chain (1:50 dilution) and anti-matrix protein gla (1:400 dilution) antibodies were purchased from Santa Cruz (Dallas, TX). The tissue inhibitor of matrix metalloproteinase 3 antibody (1:1,000 dilution) was obtained from Thermo Pierce (Rockford, IL). The anti-vascular cell adhesion protein 1 antibody (1:100 dilution) was obtained from R&D systems (Minneapolis, MN).

Monocrotaline (MCT) was purchased from Sigma-Aldrich Chemical Co., (St. Louis, MO). MCT was dissolved in 1 N HCl and at last adjusted pH to 7.4 by 1 N NaOH on each use. MCT pyrrole (MCTp) was purchased from Santa Cruz (Santa Cruz, CA) and dissolved in dimethylformamide. The stock solution of MCTp was kept at −80°C until use. Recombinant human TGF-β2 was purchased from Fujifilm Wako Pure Chemical (Osaka, Japan). Stealth RNAi of TRPM7 (M7 si1: HSS123307, M7 si2: HSS182662, M7 si3: HSS182663) and negative control siRNA were obtained from Invitrogen (Carlsbad, CA). FTY-720 was purchased from Cayman Chemical (Ann Arbor, MI) and dissolved in dimethyl sulfoxide. Antibodies against phospho-Smad2 (#3108, Cell Signaling Technology, Danvers, MA), anti-N-cadheline (#ab98952 Abcam, Cambridge, UK), anti-VE-cadheline (#ab33168 Abcam, Cambridge, UK), STAT3 (#9139, Cell Signaling Technology), phospho-STAT3 (#9138, Cell Signaling Technology), α-SMA (#ab32575, Abcam, Cambridge, UK), CD31 (#77699, Cell Signaling Technology), vWF (#ab6994, Abcam), TRPM7 (#ab85016, Abcam), Phospho-Akt (Ser473) (#4060, Cell Signaling Technology), Connexin 43 (#ab11370, Abcam) and β-actin (#ab6276, Abcam) were used for the immunoblotting and immunostaining experiments.