affinity purified polyclonal rabbit anti rat p2x 7 r  (Alomone Labs)

Journal: PLoS ONE

Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

doi: 10.1371/journal.pone.0106269

Figure Lengend Snippet: Confocal fluorescence Z stack images of flat mount bladder mucosa taken from the urothelial towards the serosal surface. (A) Granular staining for Panx1 channels (red) is observed throughout the mucosa. Staining for the intermediate filament vimentin (green) is observed on the apical urothelial region and particularly on a few cells in the lamina propria, which likely correspond to suburothelial myofibroblasts. Note partial colocalization of Pannexin 1 with vimentin-positive cells. (B) Positive staining for P2X 7 R is observed in the urothelium, blood vessels (white arrows) and lamina propria, while staining for cytokeratin 7/17 is restricted to urothelial cells. Note intense P2X 7 R immunoreactivity on the basal region of the mucosa, which is likely localized to the lamina propria myofibroblasts. DAPI nuclear staining in blue. Scale bar = 20 µm.

Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

Techniques: Fluorescence, Staining

Journal: PLoS ONE

Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

doi: 10.1371/journal.pone.0106269

Figure Lengend Snippet: Whole bladders isolated from wildtype (WT), Panx1 deficient (Panx1 −/− ) and P2X 7 R deficient (P2X 7 R −/− ) mice were bathed and instilled with PBS+glucose (1 g/L). A filling-voiding cycle was simulated by bladder instillation for 8 min at 1.5 mL/h flow rate followed by 5 min no flow, after which the bladder was emptied and ATP release in the bladder lumen was quantified. Data represent mean ± SEM (N = 9 WT, 4 Panx1 −/− and 7 P2X 7 R −/− bladders. Compared to WT: * P< 0.05 and ** P< 0.01 by Student’s t -test).

Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

Techniques: Isolation

Journal: PLoS ONE

Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

doi: 10.1371/journal.pone.0106269

Figure Lengend Snippet: Detection of Panx1 and P2X 7 R mRNA by PCR in (A) and protein by immunoblotting in (B). Total RNA from human bladder and HeLa cells were used as reference for the PCR analyses, and whole HeLa cell lysates was used as reference for immunoblotting.

Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

Techniques: Western Blot

Journal: PLoS ONE

Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

doi: 10.1371/journal.pone.0106269

Figure Lengend Snippet: (A) TRT-HU1 cells: YoPro-1 uptake induced by cell swelling (hypoosmotic shock, black line) was significantly reduced in the presence of the Panx1 channel blocker mefloquine (MFQ 100 nM; yellow line), the P2X 7 R blocker A438079 (10 µM; red line) and when both Panx1 and P2X 7 R were blocked (blue line). Except for hypoosmotic with MFQ vs. A438079 and hypoosmotic with A438079+MFQ vs. isoosmotic, all other comparisons were significantly different at time 2800 sec ( P <0.01, N≥6) by two-way repeated measures ANOVA, followed by Tukey’s multiple comparison. (B) Primary mouse urothelial cells: YoPro-1 uptake induced by hypoosmotic shock was significantly lower in P2X 7 R −/− (red line) compared to wildtype (WT) urothelial cells (black line). Dye uptake by P2X 7 R −/− cells was abolished in the presence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; green line) and was absent in Panx1 −/− urothelial cells (blue line). WT vs P2X 7 R −/− , P2X 7 R −/− +MFQ and Panx1 −/− ( P< 0.001, N = 4) by ANOVA followed by Tukey’s multiple comparison.

Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

Techniques:

Journal: PLoS ONE

Article Title: Pannexin 1 Channels Play Essential Roles in Urothelial Mechanotransduction and Intercellular Signaling

doi: 10.1371/journal.pone.0106269

Figure Lengend Snippet: (A) Mechanical stimulation imposed by rinsing the cells with bathing solution induced ATP release from TRT-HU1 cells that was significantly higher than that from non-stimulated cells when measured in the presence or absence of the Panx1 channel blocker mefloquine (MFQ, 100 nM; n = 4 each, * P <0.05 and ** P <0.01, by paired t -test.). (B) Normalized ATP release with respect to basal values, however, was significantly lower in MFQ-treated compared to untreated cells (N = 4, $ P <0.05 by Mann-Whitney U test). (C) Exposure to low divalent cation solution (LDPBS), a condition known to enhance P2X 7 R activation, significantly increased ATP release from TRT-HU1 cells. All data represent mean ± SEM (N = 4 each, * P <0.05 by Student’s t -test).

Article Snippet: The following primary antibodies were used: affinity purified polyclonal rabbit anti-rat P2X 7 R corresponding to amino acid residues 576–595 (1∶250, Alomone Labs, Jerusalem, Israel), polyclonal rabbit anti-mouse Pannexin 1 CL (Cytoplasmic loop, 1∶50, Invitrogen, Carlsbad, CA), monoclonal mouse anti-human cytokeratin 7/17 (1∶100, Santa Cruz Biotechnology, Dallas, TX) and monoclonal mouse anti-vimentin (1∶100, Sigma-Aldrich).

Techniques: MANN-WHITNEY, Activation Assay