Nf449

Two-electrode voltage clamp recordings were carried out using a GeneClamp 500B amplifier with a Digidata 1322A analog-to-digital converter and pCLAMP 8.2 acquisition software (Molecular Devices, Menlo Park, CA) at a holding potential of −60 mV. ATP (Mg²⁺ salt, Sigma) was applied via a U-tube perfusion system for 3 s at 5–10-min intervals (dependent on the P2X receptor) to allow for reproducible responses to be recorded. Antagonists were bath-perfused in ND96 buffer for 5 min before they were co-applied with an EC₉₀ of ATP through the U-tube. To generate inhibition curves, antagonists were co-applied with an EC₉₀ of ATP to standardize any shift in ATP potency. Antagonists fully equilibrated with the receptor during the first application period as the level of inhibition was maintained on a second test application. The inhibitory effects of the antagonists were reversed in the washout period between agonist applications. Antagonists (at maximal concentrations used) were applied to the WT and all mutant receptors in the absence of ATP and were seen to have no effect on the holding current. Suramin was from Sigma, PPADS from Tocris, and NF449 from Abcam.

Concanavalin A (ConA) were purchased from Sigma–Aldrich. Anti-CD3ε mAb was purchased from R&D Systems (United States). Anti-CD28 mAb was from eBioscience (United States). ATP, ADP, and adenosine were from Sigma (United States). PPADS, BDBD, MRS2578, SCH442416, PSB36, MRS3777, A438079, CGS15943, PSB603, and MRS2111 were from Tocris Bioscience (United Kingdom). Suramin and oxATP were purchased from Sigma–Aldrich. NF449 was from Abcam (United Kingdom). adenosine 5′-[γ-thio]triphosphate tetralithium salt (ATP-γ-S), α,β-methyleneadenosine 5′-triphosphate lithium salt (α,β-Me-ATP), BzATP, α,β-methyleneadenosine 5′-diphosphate sodium salt (α,β-Me-ADP), and 2-methylthioadenosine diphosphate trisodium salt (2-MeS-ADP) were purchased from Sigma–Aldrich. Anti-ERK1/2 mAb and anti-phospho-ERK 1/2 (Thr²⁰²/Tyr²⁰⁴) mAb and other secondary Abs were obtained from Cell Signaling Technology (United States). All other chemicals used were of the highest purity available.