15d pgj2

Cells seeded in 24-well plates at a density of 2 × 10⁵ cells per well and grown for 24 h were preincubated with NS-398 (1 μM) or its vehicle for 1 h. Thereafter, cells were incubated with vehicle or lovastatin lactone in the presence or absence of NS-398 for another 24 h. The final volume of the supernatant was 300 μl per well. Afterwards, cell culture media were removed and analyzed for PGE₂, PGD₂ and 15d-PGJ₂ using enzyme immunoassay kits (PGE₂, PGD₂: Cayman Chemical, Ann Arbor, MI, USA; 15d-PGJ₂: Enzo Life Sciences). For indication of percent control PG levels were normalized to whole cell protein and subsequently expressed as percent of vehicle control (100%).

Plasma were separated from normal, cocaine users, HIV positive patients, and cocaine using HIV positive subjects as previously mentioned. Plasma was analyzed for PGE₂ (GenWay Biotech Inc. San Diego, CA) and 15d-PGJ2 (Enzo Life Sciences, Farmingdale, NY) using commercially available ELISA kits as per the manufacturer’s instructions.

Fibrinogen, bovine thrombin, H89, GW9662 and IMBX were purchased from Sigma Aldrich (Poole, UK). 15dPGJ2 was purchased from Enzo Life Sciences and Ciglitazone and SQ22536 from Tocris Bioscience (Bristol, UK). The cAMP ELISA kit was from Enzo Life Sciences (Exeter, UK). Primary anti‐ FAK (focal adhesion kinase) (C20), Syk (N‐19), PLCγ2 (Q20), β3 (C20) and actin (C11) antibodies were purchased from Santa Cruz Biotechnology (Heidelberg, Germany). Phospho‐specific primary antibodies for β3 Y779 and Akt S473 were from Abcam (Cambridge, UK). Anti‐phospho–PKC (protein kinase C) substrate, phospho‐S157 and S239 VASP and phospho‐Ser 19 myosin light chain antibodies were purchased from New England BioLabs (Cell Signalling, Hitchin, UK), and anti‐phospho‐Tyr 4G10 antibody was purchased from Millipore (Watford, UK). Fluorophore conjugated secondary antibodies, Fluo‐4 calcium indicator dye and Alexa‐488 and Alexa‐647 conjugated phalloidin were purchased from Life Technologies (Paisely, UK). All other reagents were from previously described sources 26.

GW9662 (#70785) and PD146176 (#10010518) were purchased from Cayman Chemical. GW4869 (#6823-69-4) was purchased from Sigma-Aldrich. TGF-β1 (240-B-010) and IL-4 (404-ML) were purchased from R&D Systems. 15-HETE and 15d-PGJ₂ were purchased from Enzo Life Sciences. Lipoxin A4 was purchased from Neogene. Mouse IgG Blocking Reagent (MKB-2213) was purchased from Vector Laboratories.

Glenmark proprietary TRPA1 antagonists (GRC 17536, GRC 17770, GRC 17138), TRPV1 antagonist (GRC 6211) and compound 7 (TRPA1 antagonist of Janssen; WO 2009/147079) were synthesized in-house by the discovery chemistry group. A 10 mM stock of the above antagonists was prepared in DMSO. Subsequent dilutions from the stock solution were made in drug dilution buffer (DDB) (DMEM F-12 containing 1.8 mM CaCl₂). For Calcium fluorescence assay, Ca⁺², Mg⁺² free PBS were used instead of DDB for drug dilution. LPS, AITC, capsaicin, H₂O₂, and crotonaldehyde were procured from Sigma Aldrich Inc. (St Louis, MO, USA). ssRNA40 and loxoribine were purchased from Invivogen (San Diego, CA, USA), 15d-PGJ₂ from Enzo life sciences (Farmingdale, NY, USA), and citric acid from Merck (Mumbai, India).