2 mesadp

2-MeSADP, thrombin, apyrase, prostaglandin E₁ (PGE₁), sodium citrate, prednisolone, and acetylsalicylic acid (ASA) were purchased from Sigma (St. Louis, MO, USA). Anti-phospho-cPLA₂ (Ser505), anti-cPLA₂, anti-phospho-ERK (Thr202/Tyr204), and anti-total-ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). HRP-linked secondary antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Thromboxane B₂ (TxB₂) ELISA kit was purchased from Enzo Life Sciences (Exeter, UK). All other reagents were of reagent grade.

2-MeSADP, U46619, ADP, thrombin, serotonin, epinephrine, apyrase (type V), prostaglandin E₁ (PGE₁), sodium citrate, and ASA were bought from Sigma (St. Louis, MO, USA). Dr. Richard Farndale of the University of Cambridge provided CRP. AYPGKF was from Invitrogen (Carlsbad, CA, USA). Anti-phospho-Akt (Ser⁴⁷³), anti-phospho-ERK (Thr202/Tyr204), anti-Akt, anti-ERK, and anti-β-actin antibodies were from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-labeled secondary antibody was bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All additional chemicals were of reagent grade.

All reagents were purchased from Thermo Fisher Scientific unless otherwise stated. 2-MeSADP, ADP, and Apyrase (type V) were purchased from Sigma (St. Louis, MO, USA). AR-C69931MX was a gift from the Medicines Company (Parsippany, NJ, USA). AYPGKF was purchased from GenScript (Piscataway, NJ, USA). Phosphorylated Akt S473 and phosphorylated VASP S157 were purchased from Cell Signaling Technologies (Beverly, MA, USA), while the total Akt antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The total VASP antibody was purchased from Origene (Rockville, MD, USA). Odyssey blocking buffer and secondary antibodies IRDye 800CW goat anti-rabbit and IRDye 680LT goat anti-mouse were purchased from Li-Cor (Lincoln, NE, USA). Collagen and Chronolume, used for the detection of secreted ATP, were purchased from Chrono-Log Corporation (Havertown, PA, USA). GTP-bound Rap1b was assessed using a kit from Cell Signaling and cAMP production was measured using a kit from Enzo. Prostaglandin E1 was also purchased from Enzo (New York, NY, USA). AseI was purchased from New England BioLabs (Ipswitch, MA, USA).

To block the activity of P2X7Rs, either the P2X7R antagonist oxATP (100 µM, Sigma) [57 (link)] or a highly selective P2X7R antagonist A740003 (250 nM, Sigma) was used. At 250 nM, A740003 inhibits >90 % of P2X7 receptor-mediated Ca²⁺ influx [58 (link)]. To activate P2Y1Rs, the agonist 2mesADP (100 µM, Sigma) was used. At 100 µM, 2mesADP was found to excite P2Y1 receptors in inhibitory neurons in the hippocampal slice [38 (link)]. To inhibit P2Y1R activity, the potent selective P2Y1R antagonist, MRS (60 µM, Tocris) was incubated with DRGs. In neocortical subventricular zone slices, MRS, at 50 µM, effectively blocks P2Y1R-mediated Ca²⁺ responses in intermediate neuronal progenitors [59 (link)]. To study the role of p38 in P2Y1R–P2X3R control, either the p38 inhibitor, SB (100 µM, Tocris) or the p38 activator, Anis (10 nM, Sigma) was used [60 (link), 61 (link)]. The function of SGCs was disrupted by incubating DRGs in the glia Krebs cycle inhibitor, FC, (100 µM, Sigma) [62 (link)]. Unless stated otherwise, the incubation time for chemicals was 1 h.

U46619, 2-MeSADP, ADP, thrombin, serotonin, epinephrine, apyrase (type V), prostaglandin E₁ (PGE₁), sodium citrate, and acetylsalicylic acid were from Sigma (St. Louis, MO, USA). CRP was obtained from Dr. Richard Farndale at the University of Cambridge. Phycoerythrin-conjugated antibody JON/A and FITC-conjugated anti-P-selectin antibody were from Emfret Analytics (Sterntalerweg, Wurzburg, Germany). Hexapeptide AYPGKF was custom synthesized by Invitrogen (Carlsbad, CA, USA). Fura-2-AM was from Millipore (Temecula, CA, USA). Anti-phospho-Akt (Ser⁴⁷³), anti-phospho-ERK (Thr202/Tyr204), anti-phospho-PKCδ (Tyr311), anti-Akt, anti-PKCδ, and anti-ERK antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Horseradish peroxidase-labeled secondary antibody was bought from Santa Cruz Biotechnology (Santa Cruz, CA, USA). All other reagents were of analytical grade, and deionized water was used throughout the study.