Trpv1

Freshly dissected brains of WT and fads2^−/− male and female mice were mechanically homogenized in lysate buffer containing protease inhibitor cocktail (Complete; Roche). Protein concentrations were measured using a Pierce BCA protein assay kit (Thermo Scientific).
Protein aliquots (100 μg) were separated by NuPAGE 4–12% Bis-Tris gels and transferred to nitrocellulose membrane using the NuPAGE Western blot system (Invitrogen). Blots were immunostained overnight at 4°C with respective antibodies: anti-fatty acid amide hydrolase (FAAH) (1:200, #ab54615, RRID:AB_2101890) and anti-CB2 from Abcam (1:200, #ab3561, RRID:AB_303908); anti-CB1 (1:200, #sc10066, RRID:AB_637711) and anti-transient receptor potential vallinoid 1 cation channel 1 (TRPV1) from Santa Cruz (1:100, #sc398417, RRID:AB_637711); OrexinR from Alomone Labs (1:200, #AOR-001, RRID:AB_2040048); and anti-α tubulin from Sigma-Aldrich (1:12,000, #T6074, RRID:AB_477582). After washing, HRP-conjugated secondary antibodies were used and detected with the ECL system. Signals were quantified by densitometry using the ImageJ2 program (RRID:SCR_003070).

siRNA for CBS (hydroxylamine), CSE (dl-propargylglycine), TRPV1, TRPV3, TRPV6, and TRPM4 were purchased from Santa Cruz. H₂S donor NaHS was purchased from Sigma-Aldrich Corporation (St. Louis, MO, USA). Capsaicin and capsazepine were purchased from EMD Millipore (Billerica, MA, USA). The concentrations of Capsaicin and capsazepine used for the treatment were 1 μM.

Immunocytochemistry for α-SMA, vimentin, TLR4 (Abcam, Cambridge, MA) and TRPV1 (Santa Cruz, Dallas, TX) were performed on transfected myofibroblasts grown in chamber slides at several passages as previously described [5 (link)]. Cells were examined with fluorescence (TE 300 Nikon Eclipse; Nikon Instruments, Melville, NY) and confocal laser-scanning (LSM 510; Zeiss, Thornwood, NY) microscope. Fluorescent images were acquired using a 40x oil-immersion objectives.

The following antibodies were used in these studies: δ-ENaC (Lifespan Biosciences, LS-C119717) or Santa Cruz Biotechnology, goat polyclonal, sc-22246), gustducin (Santa Cruz Biotechnology, sc-395), PLCβ2 (Santa Cruz Biotechnology, sc-515912), ACE₂ (Abcam ab108252), Taste receptor type 1 member 3 (T1R3; Santa Cruz Biotechnology sc-398996), TRPV1 (Santa Cruz Biotechnology, sc 12,498 or Lifespan Biosciences, LSC172124), GPER1 (Abcam 39742), and AT1R (Millipore: AB15552). The δ-ENaC peptide was obtained from Santa Cruz Biotechnology (sc-22246P).

Western blot analysis of protein expression was performed according to Eissa and Seada [60 (link)]. Samples were electrophoretically separated on 10% gels, transferred to 0.2 µm pore-sized nitrocellulose, and incubated overnight with primary antibodies against: GPR55, PPARα (host: rabbit) β-actin (host: mouse) that were purchased from Sigma-Aldrich, (St. Louis, MO, USA). Primary antibodies against: Il-10 (host mouse), CB1, CB2, TRPV1 (host rabbit) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Primary antibodies against PPARγ (host: rat) were purchased from Abcam (Cambridge, UK). Primary antibodies against PPARδ were purchased from Invitrogen (Carlsbad, CA, USA). Primary antibodies against IL-2 (host: rat) were purchased from Thermo Fisher Scientific (Thermo Fisher Scientific, Waltham, MA, USA). Next membranes were incubated for 2 h with alkaline phosphatase secondary IgG antibody against corresponding primary antibody (Sigma-Aldrich, St. Louis, MO, USA). Protein bands were visualized using the BCIP/NBT Liquid substrate system (Sigma-Aldrich, St. Louis, MO, USA). To compare the proteins expression between samples, each band intensity was estimated using VersaDoc System and Quantity One software (Bio-Rad Laboratories Inc., Hercules, CA, USA). The results are expressed as a percentage of the expression determined in the control cells.