Tfllrn nh2

Frozen or fresh human adult lung and skin samples, ranging from 15 to 20 g, were purchased through the Collaborative Human Tissue Network; cyanogen bromide–activated Sepharose 4B from GE Healthcare; heparin-Sepharose, Na-benzoyl-dl-arginine 4-nitro-anilide, porcine pancreatic type IX trypsin (13,000–20,000 U/mg), sulphinpyrazone, calcium ionophore (A23187), porcine intestinal mucosa heparin (mean mol wt 16 kD), SBTI, cellulose phosphate (fibrous form), and leupeptin from Sigma-Aldrich; FCS, DMEM, penicillin, streptomycin, and amphotericin from Thermo Fisher Scientific; anti-PAR2 IgG and anti-EMR2 from R&D Systems; and PAR2-activating peptide (SLIGRL-NH2), PAR1-activating peptide (TFLLRN-NH2), PAR2 peptide control LRGILS-NH2, and PAR2 antagonist (FSLLRY-NH2) from Tocris Bioscience. PAR2 agonists or antagonist were diluted in a buffer comprising 20% DMSO and 80% PBS.

The V5‐tag antibody conjugated with fluorescein isothiocyanate (FITC) (catalog no. R963‐25) was from Invitrogen (Carlsbad, CA, USA). The Phospho‐p44/42 mitogen‐activated protein kinase (MAPK) (extracellular signal‐regulated kinase [ERK] 1/2) (Thr202/Tyr204) (D13.14.4E) Rabbit monoclonal antibody (mAb; catalog no. 4370) and p44/42 MAPK (ERK1/2) (3A7) Mouse mAb (catalog no. 9107) were both purchased from Cell Signaling Technology, Inc (Danvers, MA, USA). Trypsin (catalog # V511A) was from Promega (Fitchburg, WI, USA). The PAR1 activation peptide, SFLLRN‐NH₂ and TFLLRN‐NH₂ were from Tocris Bioscience (Minneapolis, MN, USA). PAR4 activation peptide, AYPGKF‐NH₂, was from GenScript (Piscataway, NJ, USA). C4a (catalog no. A106, Lot 16) was from Complement Technology (Tyler, TX, USA). Human α‐thrombin (catalog # HCT‐0020, specific activity >2989 U/mg) was from Hematological Technologies (Essex Junction, VT, USA). Vorapaxar (catalog no. 1755, batch 2) was from Axon Medchem (Reston, VA, USA). All other reagents were from Thermo Fisher Scientific (Pittsburgh, PA, USA) except where noted.