Nf157

Suramin hexasodium salt, NF023, NF110, NF157, NF279, NF340, NF449, PSB0739, and MRS2578 were purchased from Tocris Bioscience. Fondaparinux sodium (Arixtra) was purchased from GlaxoSmithKline. Pirodavir was a kind gift from Dr. John Lambert, Biota Pharmaceuticals. MRS2578 and Pirodavir were dissolved in DMSO and used for the experiments.

EpiLife culture medium with Ca²⁺ (60 μM), EpiLife defined growth supplement (EDGS), penicillin/streptomycin, and Lipofectamine 2000 reagent were purchased from Thermo Fisher Scientific (Waltham, MA USA). On-target plus siRNA was purchased from GE healthcare (Little Chalfont, UK). FNC coating mixture was purchased from Athena ES (Baltimore, MA, USA). Suramin hexasodium salt (an inhibitor of P2X and P2Y receptors), PPADS tetrasodium salt (an inhibitor of P2X and P2Y receptors), TNP-ATP triethylammonium salt (an inhibitor of P2X receptor), NF 157 (an inhibitor of P2Y₁₁ and P2X₁), ARL 67156 (an inhibitor of ecto-ATPases), and ¹⁰Panx (an inhibitor of Pannexin-1 channel) were purchased from Tocris Bioscience (Minneapolis, MN, USA). Adenosine-5’-(γ-thio)-triphosphate tetralithium salt (ATPγS, a non-hydrolyzable ATP analogue), Brilliant blue G (BBG; an inhibitor of P2X receptors), and apyrase were purchased from Sigma-Aldrich (St. Louis, MO, USA).

BBG purchased from Santa Cruz Biotechnology. ATP, ADP, UTP, ADO, CPA, H89, IBMX, BzATP, 8-Br-cAMP, Fluorescein isothiocyanate–dextran (average mol. wt. 70,000 Da) purchased from Sigma; 8-(4-Chlorophenylthio)-2′- O- methyladenosine-3′, 5′-cyclic monophosphate (8-pCPT-2′-O-Me-cAMP, 8CPT) purchased from BioLog; Forskolin (FK), CGS 15943 and NF157 purchased from Tocris Bioscience. 2′,5′-Dideoxyadenosine (ddAdo) purchased from Enzo Life Sciences. See Appendix for a more detailed list, with the concentrations used and molecular implications.

D-AP5, PPADS, suramin, Evans blue, NF157, NF546, MRS2500, MRS2365, ARL67156, GPAnt-2a and ryanodine were purchased from Tocris Bioscience (Ellisville, MO, USA). All other chemicals were obtained from Sigma-Aldrich (St. Louis, MO, USA). Adenosine, U73122, ryanodine, Bis-IV, PMA and Gö6976 were initially dissolved in DMSO for stock and then diluted in solutions to final working concentrations. The final DMSO concentration was less than 0.1%, with no effects on glycine-induced currents of GCs. All other drug solutions were prepared in ion-free water, stored at −20 °C and freshly diluted to the working concentrations using normal solutions.

Probenecid and polyoxyethylene sorbitan monolaurate (equivalent to Tween-20) were purchased from Wako Pure Chemical Industries (Osaka, Japan). 5-BDBD, AZ11645373, MRS2179, MRS2578, NF157 and MRS2211 were from Tocris Bioscience (Bristol, UK). BAPTA-AM solution was purchased from Dojindo (Kumamoto, Japan). SQ22536 was purchased from Merck (Darmstadt, Germany). Phospho-Akt rabbit monoclonal antibody (#4060), Akt rabbit monoclonal antibody (#4691), goat horseradish peroxidase (HRP)-conjugated anti-rabbit IgG antibody (#7074) and goat HRP-conjugated anti-mouse IgG antibody (#7076) were purchased from Cell Signaling Technology, Inc. (Beverly, MA). Anti-P2X7 receptor antibody (APR-004), anti-P2Y11 receptor antibody (APR-015) and anti-P2Y13 receptor antibody (APR-017) were purchased from Alomone Labs (Jerusalem, Israel). Mouse anti-β-actin antibody (sc-47778) and goat anti-rabbit IgG-FITC (sc-2012) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-connexin 43 antibody (ab11370) was purchased from Abcam (Cambridge, UK). Panx1 polyclonal antibody (12595-1-AP) was purchased from Proteintech, Inc (Chicago, IL). Unless otherwise stated, all other reagents were obtained from Sigma-Aldrich (St Louis, MO). All other chemicals used were of the highest purity available.

NF157, MRS2500, suramin, AR-C 118925XX, CGS15943, pertussis toxin (PTX), ATP, and ATPγS were from Tocris Bioscience (Bristol, UK). YM-254890 (YM) was from Wako Pure Chemical Industries (Richmond, VA). Bovine serum albumin (BSA) fraction V fatty acid-free was from Roche (Basel, Switzerland). Poly-d-lysine and apyrase were from Sigma-Aldrich (St. Louis, MO). Carbachol was from Abcam (Cambridge, UK). Triton X-100 was from Polysciences (Warrington, PA). RANTES was from Peprotech (Rocky Hill, NJ) and RANTES analog, PSC-RANTES was a gift from Oliver Hartley (Université de Genève). HEK293T cells were from American Type Culture Collection (ATCC) (Manassas, VA). Dulbecco’s Modified Eagle’s Medium GlutaMAX (DMEM), FluoroBrite DMEM, Dulbecco’s phosphate-buffered saline without Ca²⁺ and Mg²⁺ (DPBS), Hanks' Balanced Salt solution (HBSS), and HEPES buffer were from Fisher Scientific (Hampton, NH). Lipofectamine 2000 and trypsin–EDTA (0.25%, phenol red) were from ThermoFisher Scientific (Waltham, MA). Fetal bovine serum (FBS) was from Gemini Bio-Products (West Sacramento, CA). Clear and clear-bottom black 384-well microplates were from Greiner (Monroe, NC). FLIPR Calcium 6 Assay and Flexstation II 384 Plate Reader were from Molecular Devices (San Jose, CA). 384-well transfer tips for the Flexstation were from Axygen (Union City, CA).