A784168

C57Bl/6 and TRPV1 knockout (KO) mice (C57Bl/6 background) were purchased from the Jackson Laboratory (Bar Harbor, ME). The ω-hydroxylase inhibitor N-hydroxy-N’-(4-n-butyl-2-methylphenyl)formamidine (HET0016) was purchased from Enzo (Farmingdale, NY). A784168 was purchased from Tocris (Minneapolis, MN). gp91 ds-tat and gp91 ds-tat scrambled were purchased from AnaSpec (Fremont, CA). The 20-HETE stable agonist N-(20-hydroxyeicosa-5[Z],14[Z]-dienoyl)glycine (20-5,14-HEDGE) was synthesized in the laboratory of J.R. Falck.

Ethanol was purchased from American Bioanalytical (Natick, MA). A 784168 and HC 030031 were purchased from Tocris Bioscience (Bristol, United Kingdom); PKG inhibitor peptide DT-3 was purchased from Axxora LLC (Farmingdale, NY). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). For AA 784168 and HC 030031, 10 mM stock solutions were prepared in DMSO, aliquoted and stored at −20°C. On the day of the experiment, aliquots were thawed and used to prepare blocker-containing solutions at final concentration. For capsaicin-containing solutions, 1 mM capsaicin stock solution was prepared in DMSO and further diluted in PSS immediately before experiment to render the desired final concentration. For SNP and caffeine, 1 mM stock solutions were prepared in PSS daily, and further diluted in PSS to render final concentrations.

Mouse monoclonal anti-actin (AM1021b) was purchased from Abgent Biotechnology (San Diego, CA). Rabbit polyclonal anti-TRPA1 (Western blotting: PA1-46159, Invitrogen; immunofluorescence: ACC-037, Alomone). Rabbit polyclonal anti-phospho-ERKI/2 (AP3906A, Abgent), Mouse polyclonal anti-total ERK1/2 (AM2189B, Abgent), and mouse monoclonal anti-fibronectin (F6140, MilliporeSigma) were used. HRP-conjugated antirabbit and antimouse secondary antibodies were purchased from Thermo Scientific (Waltham, MA) and Abcam, respectively. Cross-adsorbed Alexa Fluor 555 goat antirabbit immunoglobulin was purchased from Life Technologies (Grand Island, NY). Highly cross-adsorbed CF555 donkey antimouse IgG (catalog # 20037) was purchased from Biotium, Inc (Fremont, CA). Unless otherwise specified, all reagents were purchased from MilliporeSigma (Burlington, MA, USA). IL-1β (ProSpec-Tany TechnoGene Ltd, East Brunswick, NJ USA) and IL-1α (Sino Biological, Life Technologies, MD, USA) were used. IL-1RA, OAG, U-0126, A-967079, and thapsigargin (Cayman Chemical; Ann Arbor, MI USA), polygodial, and A-784168 (Tocris Bioscience, Bristol, UK), BAPTA (Assay BioTech, Sunnyvale, CA USA) were used. Lyophilized IL-1β was reconstituted in PBS +0.1% BSA. Control cells were treated with PBS +0.1% BSA.