Ib meca

Adenosine, NECA, IB-MECA, HEMADO, DPCPX (8-cyclopentyl-1,3-dipropyl-7H-purine-2,6-dione) and MRS 1220 were purchased from Sigma-Aldrich and dissolved in dimethyl-sulphoxide (DMSO). CA200645, a high affinity AR xanthine amine congener (XAC) derivative containing a polyamide linker connected to the BY630 fluorophore, was purchased from HelloBio (Bristol, UK) and dissolved in DMSO. AV039, a highly potent and selective fluorescent antagonist of the human A₃R based on the 1,2,4-Triazolo[4,3-a]quinoxalin-1-one linked to BY630^{39 (link)}, was kindly gifted to us by Stephen Hill and Stephen Briddon (University of Nottingham). PMA was purchased from Sigma-Aldrich. Compounds under investigation were purchased from e-molecules and dissolved in DMSO. The concentration of DMSO was maintained to < 1.5% across all experiments (1.26% for all cAMP assays, 1% for pERK1/2 assays and 1.02% or 1.1% for NanoBRET ligand-binding experiments using CA200645 or AV039, respectively).

Monoclonal anti-ERK1/2 and anti-pERK1/2 (Thr202/Tyr204) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA); anti-F4/80 from Novus Biologicals (Littleton, CO, USA); anti-CD31, anti-VEGF-R2, anti-Bad from BD Pharmingen (San Jose, CA, USA); anti-β-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA, USA); recombinant Protein G-HRP conjugate from Thermo Scientific (Waltham, MA, USA). AOPCP (adenosine 5’-alpha,beta-methylene diphosphate), A₃AR agonist IB-MECA (N⁶-(3-Iodobenzyl)adenosine-5′-N-methyluronamide, 1-Deoxy-1-[6-[((3-Iodophenyl)methyl)amino]-9H-purin-9-yl]-N-methyl-β-D-ribofuranuronamide), A_2AAR agonist CGS-21680 (2-p-(2-Carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine hydrochloride hydrate), A₁AR agonist CCPA (2-Chloro-N⁶-cyclopentyladenosine) and cell culture reagents were purchased from Sigma-Aldrich (St Louis, MO, USA).