Sligkv nh2

The MEK inhibitor U0126 and the Rac1 inhibitor NSC23766 were purchased from Calbiochem/Merck. GB88 and GB110 were a kind gift of Dr. David B. Fairlie (The University of Queensland, Brisbane, Australia) [33 (link),34 (link)]. The PAR2-selective peptide agonist SLIGKV-NH₂ and the PAR1-selective agonist peptide TFLLRN-NH₂ (STRAP-1) were obtained from Bachem (Bubendorf, Switzerland). Synthesis, coupling, cleavage from the resin and characterization of the PAR2-selective peptide 2-furoyl-LIGRLO-NH₂ (2f-LI, EC₅₀ = 2.5 μM) were done as described in detail before [26 (link)]. The following primary antibodies were used: anti-ALK5 antibody (TGFβ RI (V22), Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho-ERK1/2 (#4370, Cell Signalling Technology, Frankfurt/Main, Germany), anti-HSP90 (both #sc-7947 and #sc-13119), anti-ERK1/2 (#AF1576, R&D Systems, Wiesbaden, Germany), and anti-HA (clone 12CA5, Roche, Mannheim, Germany). HRP-linked anti-rabbit (#7074), anti-mouse (#7076), and anti-rat (#7077) secondary antibodies were from Cell Signaling Technology. The rhTGF-β1 (#300-023) was purchased from ReliaTech (Wolfenbüttel, Germany) and used at a concentration of 5 ng/mL.

Prior to determination of proliferation, HCSMC were starved for 24 h. Cells were pre-incubated with the specific PAR2 antagonist GB83 (10 μM, Axon Medchem, dissolved in DMSO), the vascular endothelial growth factor receptor 2 neutralization antibody (VEGFR2-NA, R&D Systems Wiesbaden-Nordenstadt, Germany, MAB3572) or calcium dobesilate (Sigma-Aldrich, Schnelldorf, Germany) for 1 h. Subsequently, cells were treated either with CM alone, CM in combination with GB83, the PAR2 activating peptide (PAR2-AP, SLIGKV-NH₂; 50 μM, Bachem, Bubendorf, Switzerland), or PAR-AP in combination with the VEGFR2-NA or dobesilate for 24 h. Five percentage FCS was used as a positive control. All treatments contained 10% BrdU. Proliferation was assessed by measuring BrdU incorporation (Proliferation Assay, Roche, Mannheim, Germany) with a microplate reader (Infinite M200, Tecan GmbH, Männersdorf, Switzerland).