Sligkv nh2

Manufactured by Bio-Techne

Sourced in United Kingdom

SLIGKV-NH2 is a synthetic peptide. It functions as a selective agonist for the protease-activated receptor 2 (PAR2).

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6 protocols using sligkv nh2

Modulation of Protease-Activated Receptors

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The PAR-1 inhibitor vorapaxar (Axon Medchem, Reston, VA, USA), PAR-1 agonist SFLLRN-NH2 (American Peptide Company, Sunnyvale, CA, USA), PAR2 agonists 2f-LIGRL-NH2 (EMD Millipore, Billerica, MD, USA), SLIGKV-NH2 (Tocris, Minneapolis, MN, USA), AC55541 (Tocris), TPCK-treated bovine trypsin (10,000 Nα-benzoyl-l-arginine ethyl ester units/mg, Sigma, St Louis, MO, USA), and human thrombin (1000 NIH units/mg; Sigma) were resuspended following the manufacturer’s instructions. Tryptase purified from human mast cells (70 units/mg, Fitzgerald, Acton, MA, USA) mixed with 15 kDa heparin from porcine stomach (Sigma) in a 1:10 molar ratio of tryptase to heparin immediately after thawing,^{38 (link)} and PAR2 inhibitor ENMD-1068 (50 μg/ml; Enzo Life Sciences, Farmingdale, NY, USA) were used as previously described.^{39 (link)} Tosyl-l-lysyl-chloromethane hydrochloride (TLCK; Sigma), an irreversible inhibitor of trypsin and trypsin-like serine proteases, was used at 10 nM, and the PAR-1 inhibitor vorapaxar was used at 25 μg/ml, both in RPMI/2% BSA.

White M.J., Chinea L.E., Pilling D, & Gomer R.H. (2017). Protease activated‐receptor 2 is necessary for neutrophil chemorepulsion induced by trypsin, tryptase, or dipeptidyl peptidase IV. Journal of Leukocyte Biology, 103(1), 119-128.

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Molecular Mechanisms of G Protein-Coupled Receptor Signaling Regulation

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Primary antibodies and their sources were as follows: COX-2 (Cell Signaling Technologies (CST) 12282), β-actin (CST 3700), P-ERK (CST 4370), T-ERK (CST 9102), cPLA₂ (CST 2832), STIM1 (Feske Lab #3917), α-Tubulin (Abcam ab52866). Pharmacological tools used in the study were: UTP (Sigma U6875), SLIGKV-NH2 (Tocris 4153), FK-506 (Tocris 3631), BTP2 (Sigma 203890), ATP (Sigma A6419), DiclofeNAC (Tocris 4454), Apocynin (Tocris 4663), NAC (Sigma 106425), U0126 (Tocris 1144), ATPγS (Tocris 4080), AACOCF3 (Tocris 1462), AR-C 118925XX (Tocris 4890), NF546 (Tocris 3892), S3QEL 2 (Tocris 5735), ADPβS (Sigma A8016), UDP (Sigma U4125), NF157 (Tocris 2450), Apyrase (Sigma A6410 Grade VI, High ATPase/ADPase activity), TNP-ATP (Tocris 2464), 5-BDBD (Tocris 3579), A740003 (Tocris 3701), Suramin (ACROS Organics-Fisher AC328540500), PPADS (Tocris 0625), CM4620 was a kind gift from CalciMedica.

Kountz T.S., Jairaman A., Kountz C.D., Stauderman K.A., Schleimer R.P, & Prakriya M. (2021). Differential regulation of ATP- and UTP-evoked PGE2 and IL-6 production from human airway epithelial cells. Journal of immunology (Baltimore, Md. : 1950), 207(5), 1275-1287.

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Characterization of Ovarian Cancer Cell Lines

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A2780 cell line was purchased from Sigma-Aldrich. 2008 cell line was kindly provided by Dr. François X. Claret (University of Texas M. D. Anderson Cancer Center). RMG-1 and HCH-1 were a kind gift of Dr. Hiroaki Itamochi (Tottori University, Japan). All other cell lines were purchased from the American Type Culture Collection and were maintained in either DMEM (SKOV-3, OVCAR-3, OVCAR-8, CAOV-3 and IGROV-1), McCoy's 5a Medium Modified (HT-29 and ES-2) or RPMI-1640 (2008, RMG-1, ECC-1 and HCH-1), supplemented with 10% fetal calf serum (or 20% for OVCAR-3), penicillin (100 units/mL), and streptomycin (100 μg/mL) at 37°C with 5% CO2 in a humidified incubator. Reagents were obtained as follows: Trypsin (Sigma-Aldrich cat. #: T1426). HE4 (Fujirebio); SLIGKV-NH₂ (Tocris cat. #: 3010); A2M (Prospec cat. #: PRO-551); TATI (R&D systems cat. #: 7496-PI); SLPI (R&D systems cat. #: 1274-PI); PAR-2 Antibody (Santa Cruz Biotechnology cat. #: sc-13504); ERK and phospho-ERK antibodies (Cell Signaling Technology cat. #: 9102 and 4370 respectively).

Kim K.K., Turner R., Khazan N., Kodza A., Jones A., Singh R.K, & Moore R.G. (2020). Role of trypsin and protease-activated receptor-2 in ovarian cancer. PLoS ONE, 15(5), e0232253.

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Reagent Sourcing for Biochemical Assays

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Cry j1 was purchased from Hayashibara Co., Ltd (Okayama, Japan), thrombin from Nakarai Tesque, Inc. (Tokyo, Japan), soybean trypsin inhibitor (SBTI) from Sigma-Aldrich (St. Louis, MO, USA), bivalirudin from ProSpec-Tany TechnoGene Ltd. (Rehovot, Israel), tranexamic acid from LKT Laboratories, Inc. (St. Paul, MN, USA), PAR-1 agonist TFLLR-NH2 from Tocris Bioscience (Bristol, UK), PAR-2 agonist SLIGKV-NH2 from Tocris Bioscience, Nonidet P-40 (MP40) from Nakarai Tesque, Inc. (Tokyo, Japan), and protein inhibitor cocktail from Sigma-Aldrich (St. Louis, MO, USA).

Nakanishi S., Kumamoto J, & Denda M. (2018). Tranexamic acid blocks the thrombin-mediated delay of epidermal permeability barrier recovery induced by the cedar pollen allergen, Cry j1. Scientific Reports, 8, 15610.

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Detailed Pharmacological Reagents Protocol

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AngII, poly-L-ornithine, arginine–vasopressin, Bradykinin and Isoproterenol were from Sigma. Prostaglandin F2α was from Cayman Chemical (Ann Arbor, MI). [Sar¹, Ile⁸]-AngII (SI) and [Asp¹, Val⁵, Gly⁸]-AngII (DVG) were synthesized at the Université de Sherbrooke (Canada, QC). SR121463B was kindly provided by Sanofi Aventis (Bridgewater, NJ). DCPMP was synthetized at the medicinal chemistry platform of the Institute for Research in Immunology and Cancer (IRIC, Montreal, Canada). SLIGKV-NH₂ and salbutamol were from Tocris Bioscience (Bristol, United Kingdom). Iodine-125 was obtained from PerkinElmer. Dulbecco's modified Eagles medium (DMEM), fetal bovine serum (FBS) and other cell culture reagents were purchased from Invitrogen. Prolume Purple (Methoxy e-CTZ), coelenterazine 400a and coelenterazine-h were purchased from Nanolight Technology and Goldbio. Phusion DNA polymerase was from Thermo Scientific. Restriction enzymes and T4 DNA ligase were obtained from NEB. HTS was performed at the IRIC (Université de Montreal) using 1,260 compounds from Microsource Discovery Spectrum library (msdiscovery.com).

Namkung Y., Le Gouill C., Lukashova V., Kobayashi H., Hogue M., Khoury E., Song M., Bouvier M, & Laporte S.A. (2016). Monitoring G protein-coupled receptor and β-arrestin trafficking in live cells using enhanced bystander BRET. Nature Communications, 7, 12178.

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Investigating STAT1-IRF1 Signaling Cascades

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Primary antibodies were as follows: P-STAT1 (CST 7649), β-actin (CST 3700), T-STAT1 (CST 9172), IRF1 (CST 8478), P-TBK1 (CST 5483), IRF3 (CST 11904), p65 (CST 8242), LaminA/C (CST 4777), T-TBK1 (Abcam ab40676), α-Tubulin (Abcam ab52866). Pharmacological tools were as follows: UTP (Sigma U6875), SLIGKV-NH2 (Tocris 4153), ATP (Sigma A6419), Adenosine (Tocris 3624), AR-C 118925XX (Tocris 4890), histamine (Tocris 3545), BTP2 (Sigma 203890), cetirizine (Tocris 2577), YM-254890 (Cayman Chemical 29735), Gö 6983 (Tocris 2285), GF 109203X (Tocris 0741), PDBu (Tocris 4153), PMA (Tocris 1201), ruxolitinib (Tocris 7054), ARL 67156 (Tocris 1283), NECA (Tocris 1691), IFN-β (R & D systems 8499-IF), poly(I:C) (Invivogen tlrl-picw), 2,3 cGAMP (Invivogen tlrl-nacga23), ISD (Invivogen tlrl-isdn), 3p-hpRNA (Invivogen tlrl-hprna).

Kountz T.S., Biyasheva A., Schleimer R.P, & Prakriya M. (2022). Extracellular Nucleotides and Histamine suppress TLR3- and RIG-I mediated release of antiviral Interferons from Human Airway Epithelial Cells. Journal of immunology (Baltimore, Md. : 1950), 208(10), 2390-2402.

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