Fig 2: Immunoprecipitation with anti-P2X1 antibody. Antibodies to P2X1 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads were resolved by SDS-PAGE, and Western blots were probed with A) P2X1, B) P2X4 or C) Nt5e antibodies. (A) Left and right panels show P2X1 immunoblots on IP and FT lysates from wild type and P2X4-/- mice. Monomeric P2X1 can be seen highly concentrated in the pulldown fraction at 50 kDa. Little P2X1 appears in FT. (B) P2X4 antibody detects P2X4 as a band at 70 kDa in wild type, but is completely absent in P2X4-/- mice. The antibody shows minor cross-reactivity to possibly P2X1. Note, there is no evidence of the 70 kDa band in the IP lane. (C) An antibody to 5'-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X1 is ‘clean’ with no non-specific protein binding evident in the IP lanes.

Fig 3: The Piezo1 agonist Yoda1 induced increases in [Ca2+]i in platelets and Meg-01 cells. A–C, [Ca2+]i responses to Yoda1 (25 µm) assessed in stirred Fura-2-loaded washed suspensions of platelets (top panels) and Meg-01 cells (bottom panels). A and B show representative recordings, and C shows the average peak [Ca2+]i increases (n = 4) for Yoda1 in the presence of extracellular Ca2+ compared with its vehicle control (DMSO) and following removal of external Ca2+ (EGTA). D, representative intracellular Ca2+ recording (Fluo-3 F/F0 fluorescence) from a single platelet attached to a PECAM-1-coated glass coverslip in the presence of Yoda1 and exposed to two cycles of no flow (white regions) and arterial shear (gray regions). See Fig. 2C, (panel i) for the control trace. E, average Ca2+ increases above baseline (F/F0·4 min) in the presence and absence of Yoda1 under conditions of no flow and normal arterial shear (n = 20, 35, 20, and 35 cells in HBSS no flow, Yoda1 no flow, HBSS normal arterial, and Yoda1 normal arterial conditions, respectively). **, p < 0.01; †, p < 0.01 compared with no flow, in the presence of HBSS; ‡, p < 0.001 compared with no flow in presence of Yoda1. Apyrase was omitted from the extracellular buffer to avoid P2X1 receptor responses (see Fig. 8).

Fig 4: Immunoprecipitation with anti-P2X4 antibody. Antibodies to P2X4 were immobilized onto resin beads and then incubated with mouse bladder lysates to IP the antigen and co-IP interacting proteins. Proteins that were bound (IP: 2.5 µg protein/lane) or did not bind (FT: 25 µg protein/lane) to the beads, were resolved by SDS-PAGE, and Western blots were probed with A) P2X1, B) P2X4 or C) Nt5e antibodies. (A) Left and right panels show P2X1 immunoblots on IP and FT lysates from wild type and P2X4-/- mice. P2X1 is highly concentrated in the FT fractions. Minor potential P2X1 staining appears in the IP lane, however this is due to P2X4 antibody cross-reacting and pulling down some P2X1. (B) P2X4 antibody detects P2X4 as a band at 70 kDa in wild type IP lane, but is absent in P2X4-/- mice. The antibody shows minor cross-reactivity to P2X1 (50 kDa band). (C) An antibody to 5'-nucleotidase (Nt5e) demonstrates that pulldown with anti-P2X4 is ‘clean’ with no non-specific protein binding evident in the IP lanes.

Fig 5: Localization of P2X receptor subunits on astroglia. Localization of P2X receptors in astroglia was characterized by double immunofluorescence staining of P2X1 (A), P2X2 (B), P2X3 (C), P2X4 (D), P2X5 (E), P2X6 (F), P2X7 (G) using their respective Alomone Labs antibodies (Cy3, red) and GFAP (Cy2, yellow green). Strong staining for the P2X1 receptor was detected only in axon-like fibers (A). No clear immunoreactivity was observed for P2X2 and P2X3 receptor subtypes on astroglia (B, C). P2X4, P2X5, P2X6, P2X7 receptors were found to be expressed (Da, Ec, Fa, Ga). As representative control images are the anti-P2X5 receptor antibody (#APR-005) pre-incubated with its control peptide antigen (Ea) and GFAP fluorescence alone (Ea–d). Adapted from reference 65 with permission of Elsevier