Rgd peptide

To determine activities of MMP2 and MMP9, 3 × 10⁶ cells were plated into 15-cm dishes in OFCS. After 16 h, cells were incubated with 1 μM RGD peptide (Santa Cruz Biotechnology) or vehicle (DMSO). After 72 h, supernatants were collected and cleared of cells and debris at 500 g for 10 min and 5,500 g for 20 min, respectively, using an Allegra 25R centrifuge (Beckman Coulter). Subsequently, supernatants were concentrated at 5,500 g on VivaSpin 20 columns (GE Healthcare Life Sciences) for 30 min. Concentrated samples were diluted 1:1 in Tris-glycine SDS gel loading buffer (Novex, Invitrogen) and separated by electrophoresis on 10% Tris-glycine gels containing 0.1% gelatine (Novex, Invitrogen) at 125 Volts for 90 min. Gels were incubated in 1× Zymogram renaturation and developing buffer (Novex, Invitrogen) according to the manufacturer's specification. Subsequently, gels were stained with SimplyBlue SafeStain (Novex, Invitrogen) for 6–12 h with 3–5 changes until protein bands were clearly visible.

Recombinant human TGF-β1 was purchased from PeproTech (Princeton, NJ, USA). SB431542 was purchased from Selleckchem (Houston, TX, USA). RGD peptide was purchased from Santa Cruz Biotechnology (Dallas, TX, USA). TGF-β neutralizing antibody was purchased from R&D Systems (Minneapolis, MN, USA). Anti-TNC antibodies for IHC staining and western blot analysis were purchased from Santa Cruz Biotechnology and Abcam (Cambridge, UK), respectively. Anti-GAPDH antibody was from AbClone (Seoul, Korea). Anti-SMAD2 (SMAD family member 2), anti-phospho-SMAD2, anti-SMAD3, and anti-phospho-SMAD3 antibodies were from Cell Signaling Technology (Danvers, MA, USA). Anti-pro-COL1A1 and anti-MMP-1 antibodies have been described previously [49 (link)]. Horseradish peroxidase (HRP)-conjugated goat anti-mouse IgG, goat anti-rabbit IgG, and rabbit anti-goat IgG antibodies were purchased from KOMA Biotech (Seoul, Korea). Rhodamine Red-X-conjugated goat anti-mouse IgG and Alexa Fluor 488-conjugated rabbit anti-goat IgG antibodies were purchased from Invitrogen (Carlsbad, CA, USA).

Anti-FAK, anti-Pyk2, the anti-phosphotyrosine antibody, 4G10, and HRP-conjugated goat anti-mouse and mouse anti-rabbit light chain specific IgGs were all obtained from Millipore (Lake Placid, NJ, USA); normal rabbit and mouse IgGs and RGD peptide were from Santa Cruz (CA, USA), while anti-PLCγ2 and anti-Hic-5 were from Cell Signaling Technology, Inc. (Boston, MA, USA). Anti-Rac1 was from Tebu-Bio (Peterborough, UK). Cross-linked collagen related peptide (CRP) was purchased from Prof. Richard Farndale (Dept of Biochemistry, Cambridge University, UK). The pharmacological inhibitors, PF-573228 (hereafter referred to as PF-228), PP2, Wortmannin, EHT-1864, U73122, GF109302× and the Ca²⁺ chelator, BAPTA, were from Tocris Bioscience (R&D Systems Europe, UK). Tyrphostin A9 was from Calbiochem. ML171 (2-acetylphenothiazine), and BAY61-3606 (hereafter referred to as BAY) were purchased from Sigma Aldrich (St. Louis, MO, USA).