Journal: Disease Markers

Article Title: Schisandrin B Alleviates Diabetic Cardiac Autonomic neuropathy Induced by P2X7 Receptor in Superior Cervical Ganglion via NLRP3

doi: 10.1155/2023/9956950

Figure Lengend Snippet: Effect of Schisandrin B on expression levels of P2X7 receptor in SCG of DM rats. (a) The schedule of experiment process. (b) The protein level of P2X7 protein in all group was measured by Western blotting. (c) The relative expression level of P2X7 protein to individual β -actin internal control was shown. Data are mean ± SD from four independent experiments. ( ∗ P < 0.05, ∗∗ P < 0.01 compared with the control group; # P < 0.05 compared with the DM group).

Article Snippet: After 30-min incubation with goat serum blocking solution, the sections were incubated with mouse anti-GS (1 : 150, ab64613; Proteintech) and rabbit anti-P2X7 (1 : 150, APR-002; alomone labs) overnight at 4°C in cassette, followed by rinsing and incubating sections with TRITC-labeled goat anti-rabbit IgG (1 : 150, E031320-01; EARTHYX) or FITC-labeled goat anti-mouse IgG (1 : 150, E031210-01; EARTHYX) for 1 h at 37°C.

Techniques: Expressing, Western Blot

Journal: Disease Markers

Article Title: Schisandrin B Alleviates Diabetic Cardiac Autonomic neuropathy Induced by P2X7 Receptor in Superior Cervical Ganglion via NLRP3

doi: 10.1155/2023/9956950

Figure Lengend Snippet: Effect of Schisandrin B on heart rate and blood pressure in rats.

Article Snippet: After 30-min incubation with goat serum blocking solution, the sections were incubated with mouse anti-GS (1 : 150, ab64613; Proteintech) and rabbit anti-P2X7 (1 : 150, APR-002; alomone labs) overnight at 4°C in cassette, followed by rinsing and incubating sections with TRITC-labeled goat anti-rabbit IgG (1 : 150, E031320-01; EARTHYX) or FITC-labeled goat anti-mouse IgG (1 : 150, E031210-01; EARTHYX) for 1 h at 37°C.

Techniques: shRNA

Journal: Disease Markers

Article Title: Schisandrin B Alleviates Diabetic Cardiac Autonomic neuropathy Induced by P2X7 Receptor in Superior Cervical Ganglion via NLRP3

doi: 10.1155/2023/9956950

Figure Lengend Snippet: Effects of Schisandrin B on heart rate variability in rats.

Article Snippet: After 30-min incubation with goat serum blocking solution, the sections were incubated with mouse anti-GS (1 : 150, ab64613; Proteintech) and rabbit anti-P2X7 (1 : 150, APR-002; alomone labs) overnight at 4°C in cassette, followed by rinsing and incubating sections with TRITC-labeled goat anti-rabbit IgG (1 : 150, E031320-01; EARTHYX) or FITC-labeled goat anti-mouse IgG (1 : 150, E031210-01; EARTHYX) for 1 h at 37°C.

Techniques: shRNA

Journal: Disease Markers

Article Title: Schisandrin B Alleviates Diabetic Cardiac Autonomic neuropathy Induced by P2X7 Receptor in Superior Cervical Ganglion via NLRP3

doi: 10.1155/2023/9956950

Figure Lengend Snippet: The double-label immunofluorescence staining of P2X7 and GS in SCG. The expression of P2X7 receptor and GS in SCG was detected by double-label immunofluorescence staining. The green signal represents GS staining with FITC, and the red signal indicates P2X7 staining with TRITC. The merged image represents the double staining of P2X7 and GS. Scale bar, 20 μ m.

Article Snippet: After 30-min incubation with goat serum blocking solution, the sections were incubated with mouse anti-GS (1 : 150, ab64613; Proteintech) and rabbit anti-P2X7 (1 : 150, APR-002; alomone labs) overnight at 4°C in cassette, followed by rinsing and incubating sections with TRITC-labeled goat anti-rabbit IgG (1 : 150, E031320-01; EARTHYX) or FITC-labeled goat anti-mouse IgG (1 : 150, E031210-01; EARTHYX) for 1 h at 37°C.

Techniques: Immunofluorescence, Staining, Expressing, Double Staining

Journal: The Journal of Neuroscience

Article Title: Dual Role of the P2X7 Receptor in Dendritic Outgrowth during Physiological and Pathological Brain Development

doi: 10.1523/JNEUROSCI.0805-22.2022

Figure Lengend Snippet: Neurons from P2rx7 −/− mice present less dendritic outgrowth. A , Scheme of the in vitro experiment. B , Immunostaining of a neuronal marker (MAP2) and an astroglial marker (GFAP) and of a transfection marker (eGFP) and a neuronal marker (MAP2) as the control for the transfection in a representative control WT primary hippocampal neuron at DIV10. Nuclei were counterstained with Hoechst. Scale bar, 200 µm. C , Representative hippocampal neurons from WT and P2X7R KO mice transfected with and stained for eGFP. D , Sholl analysis showing the number of intersections versus distance from the soma (radius, μm). E , Using HPLC, nucleotide concentrations in the supernatants of WT and KO primary hippocampal neurons were measured. Quantification of the ( F ) cell body area, ( G ) number of primary dendrites, ( H ) number of endings, and ( I ) mean dendritic length. ** p < 0.005. **** p < 0.0001. ns, non-significant. Data presented as Mean±SEM.

Article Snippet: For P2X7R staining, cells were incubated with a rabbit α-P2X7 antibody (Alomone Labs, RRID: AB_2040068 ) diluted 1:100 for two nights and then with AlexaFluor-549-conjugated anti-rabbit (Invitrogen, RRID: AB_2762824 ) and AlexaFluor-488-conjugated anti-rabbit (Invitrogen, RRID: AB_2633280 ) secondary antibodies diluted 1:500.

Techniques: In Vitro, Immunostaining, Marker, Transfection, Staining

Journal: The Journal of Neuroscience

Article Title: Dual Role of the P2X7 Receptor in Dendritic Outgrowth during Physiological and Pathological Brain Development

doi: 10.1523/JNEUROSCI.0805-22.2022

Figure Lengend Snippet: Pyramidal neurons from P2rx7 −/− mice present less dendritic outgrowth in the CA1 region of the hippocampus. A , Representative pyramidal hippocampal neurons in slices of the hippocampal CA1 region from P20-29 WT and P2X7R KO mice. B , Sholl analysis showing the number of intersections versus distance from the soma for WT and KO pyramidal neurons. Quantification of the ( C ) cell body area, ( D ) number of primary dendrites, ( E ) number of endings, and ( F ) mean dendritic length. G , Representative pyramidal hippocampal neurons in slices of the hippocampal CA3 region from P20-29 WT and KO mice. H , Sholl analysis showing the number of intersections versus distance from the soma for WT and KO pyramidal neurons. Data are mean ± SEM and analyzed using two-way ANOVA followed by Bonferroni's multiple comparison test. Quantification of the ( I ) cell body area, ( J ) number of primary dendrites, ( K ) number of endings, and ( L ) mean dendritic length. Morphologic deficits correlated with observed deficits in cognitive performance in younger animals. Different aspects of episodic memory, such as ( M ) OL and ( N ) TOR, were analyzed. Deficits in cognitive performance were observed according to both ( O ) the OL index (%) and ( P ) the TOR index (%). Q , Scheme of the experiment. NOR was analyzed ( R ) 2 min after the exploration phase and ( S ) after a longer delay of 24 h. ** p < 0.005. **** p < 0.0001. * p < 0.05. ns, non-significant. Data presented as Mean±SEM.

Article Snippet: For P2X7R staining, cells were incubated with a rabbit α-P2X7 antibody (Alomone Labs, RRID: AB_2040068 ) diluted 1:100 for two nights and then with AlexaFluor-549-conjugated anti-rabbit (Invitrogen, RRID: AB_2762824 ) and AlexaFluor-488-conjugated anti-rabbit (Invitrogen, RRID: AB_2633280 ) secondary antibodies diluted 1:500.

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: 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

Journal: PLoS ONE

Article Title: Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220 + Double-Negative T Lymphocytes of Autoimmune MRL/ lpr Mice

doi: 10.1371/journal.pone.0052161

Figure Lengend Snippet: Spleen cells from 3 to 4-mo-old MRL +/+ ( solid line ), MRL/ lpr ( dashed line ) and P2X7-deficient B6 P2X7R−/− ( dotted line ) mice (n = 3 mice/group) were treated for 45 min at 37°C with doses of ATP ranging from 100 to 5000 µM. Spleen cells were then triple-stained with anti-CD90, anti-CD19 and anti-CD62L mAb to assess by flow cytometry: (A) the percentage of CD62L-expressing CD19 – CD90 + T cells and (B) MFI of CD62L on CD19 – CD90 + T cells. Results are expressed as the mean percentage of initial expression ± SE.

Article Snippet: The membranes were blocked in TBS containing 3% nonfat dry milk and 0.2% Tween 20 for 1 h at room temperature and subsequently incubated overnight at 4°C with affinity purified rabbit anti-P2X7R antibodies recognizing residues 576 to 595 of P2X7R (1∶1000; Alomone Laboratories, Jerusalem, Israel).

Techniques: Staining, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220 + Double-Negative T Lymphocytes of Autoimmune MRL/ lpr Mice

doi: 10.1371/journal.pone.0052161

Figure Lengend Snippet: (A) The levels of CD39 expression on B220 – (either CD4 + or CD8 + ) (□) and B220 + DN (▪) T-cell subsets as well as on CD19 + B cells (▪) from MRL/ lpr mice (n = 3) were analyzed by flow cytometry. Results shown on histogram are normalized MFI (nMFI) calculated as MFI of positive cells × percentage of positive cells. Asterisks denote statistically significant differences between B220 + DN T cells or B220 – CD8 + T cells and B220 – CD4 + T cells: * p ≤0.05; ** p ≤0.01. (B) P2X7R mRNA expression was analyzed in FACS-purified B220 – (□) and B220 + (▪) CD19 – CD90 + T-cell subsets from individual B6, MRL +/+ and MRL/ lpr mice by real-time RT-PCR. Specific P2X7R cDNA was quantified by standard curves based on known amounts of PCR-amplified P2X7R cDNA. Data are expressed as amount (pg) of P2X7R RNA per cell, and histograms represent the mean ± SE of three independent experiments (n = 8 mice/group). (C) P2X7R protein levels in whole LN cells from 3 to 4-mo-old MRL +/+ , MRL/ lpr and B6 P2X7−/− mice, and FACS-sorted B220 – and B220 + CD19 – CD90 + T-cell subsets from MRL/ lpr mice were analyzed by western blotting using affinity-purified rabbit anti-P2X7R polyclonal antibodies (1∶1000; Alomone Laboratories). A non-specific band (*) is frequently observed with this polyclonal antibody . The blot was stripped and reprobed with anti-actin mAb. (D and E) Flow cytometric analysis of P2X7R expression levels on B220 – and B220 + T-cell subpopulations. Spleen cells from MRL +/+ , MRL/ lpr and B6 P2X7R−/− mice (n = 3 mice/group) were stained with anti-CD90 mAb, anti-B220 mAb, rabbit polyclonal anti-P2X7R antiserum (1∶100) generated by genetic immunization and with either anti-CD4 mAb, anti-CD8 mAb or anti-CD4 plus anti-CD8 mAbs. (D) Overlay histograms showing the expression levels of P2X7R on gated CD4 T cells or CD8 T cells from MRL +/+ mice (open histogram) and B6 P2X7R−/− mice (shaded histogram). (E) Overlay histograms comparing the expression levels of P2X7R on B220 – (–) and B220 + (–) T cells, either CD4 + ( left ) or CD8 + ( middle ), from MRL/ lpr mice. Right , overlay histogram comparing the expression levels of P2X7R on B220 + DN T cells (–) and B220 – CD4 + T cells (–) from MRL/ lpr mice. The results shown are representative of at least four independent experiments.

Article Snippet: The membranes were blocked in TBS containing 3% nonfat dry milk and 0.2% Tween 20 for 1 h at room temperature and subsequently incubated overnight at 4°C with affinity purified rabbit anti-P2X7R antibodies recognizing residues 576 to 595 of P2X7R (1∶1000; Alomone Laboratories, Jerusalem, Israel).

Techniques: Expressing, Flow Cytometry, Purification, Quantitative RT-PCR, Amplification, Western Blot, Affinity Purification, Staining, Generated

Journal: Mediators of Inflammation

Article Title: Macrophage P2X7 Receptor Function Is Reduced during Schistosomiasis: Putative Role of TGF- β 1

doi: 10.1155/2014/134974

Figure Lengend Snippet: Intracellular Ca 2+ measurement (using Fura-2-AM) in macrophages subjected to treatment with the P2X7 agonist BzATP and/or with the antagonist A740003. (a) Changes in intracellular Ca 2+ levels in macrophages from control (black line) and S. mansoni -infected (red line) mice. Inset: representative images from macrophages (control) before (baseline) and after treatment with 100 μ M BzATP. (b) Changes in intracellular Ca 2+ levels ([Ca 2+ ] i ) in macrophages from control (white bars) or S. mansoni- infected (black bars) mice. Three plates were used for each condition/animal, 10 cells/plate were chosen randomly for imaging, and cells were obtained from three animals. Data were expressed as mean and SEM. * P < 0.05; *** P < 0.001 (one-way ANOVA followed by post hoc Newman-Keuls test).

Article Snippet: Membranes were blocked for 1 h in Tris-buffered saline (TBS) with 2% nonfat milk and then incubated overnight with rabbit anti-P2X7 receptors (APR-004; Alomone Labs, Israel; 1 : 200; Peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acid residues 576–595 of P2X7 receptor intracellular, C-terminus) or mouse monoclonal anti- β -actin (1 : 20.000) primary antibodies.

Techniques: Infection, Imaging

Journal: Mediators of Inflammation

Article Title: Macrophage P2X7 Receptor Function Is Reduced during Schistosomiasis: Putative Role of TGF- β 1

doi: 10.1155/2014/134974

Figure Lengend Snippet: P2X7 receptors expression in peritoneal macrophages. (a) Macrophages from uninfected (white bar) and S. mansoni -infected (black bar) mice express similar levels of P2X7 protein. n = 9 replicates from three different animals, for each group. The images above are representative Western blots (U = uninfected and I = infected). (b) P2X7 receptors expression in peritoneal macrophages from control mice (white bar, untreated) or TGF- β 1-treated cells (5 ng/mL, 24 h; black bar) are also similar. The images above are representative Western blots (U = uninfected; TGF- β 1= TGF- β 1-treated macrophages). Lysates from P2X7 KO macrophages were used as negative controls for P2X7 protein expression. Cells lysates were obtained from three animals for each group. (c) Quantitative RT-PCR (qRT-PCR) analysis of P2X7 receptor mRNA levels in peritoneal macrophages. Similar levels of P2X7 mRNA were found in uninfected (white bar), S. mansoni -infected mice (black bar), and TGF- β 1-treated (5 ng/mL, 24 h; hatched bar) mouse macrophages. N = 3–5 samples per group (using different animals).

Article Snippet: Membranes were blocked for 1 h in Tris-buffered saline (TBS) with 2% nonfat milk and then incubated overnight with rabbit anti-P2X7 receptors (APR-004; Alomone Labs, Israel; 1 : 200; Peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acid residues 576–595 of P2X7 receptor intracellular, C-terminus) or mouse monoclonal anti- β -actin (1 : 20.000) primary antibodies.

Techniques: Expressing, Infection, Western Blot, Quantitative RT-PCR

Journal: Mediators of Inflammation

Article Title: Macrophage P2X7 Receptor Function Is Reduced during Schistosomiasis: Putative Role of TGF- β 1

doi: 10.1155/2014/134974

Figure Lengend Snippet: Confocal microscopy analysis of P2X7 receptor expression on the cell surface of macrophages. Cells were labelled with antibodies recognizing P2X7 receptors (green) and F4/80+ (red) as well as with DAPI (blue). (a) Representative images showing macrophages from uninfected (left column), S. mansoni -infected (middle column), and TGF- β 1-treated (right column; 5 ng/mL, for 24 h) mice. The bottom row shows orthogonal slices of the representative labelled cells, with red arrows marking P2X7 expression on the plasma membrane. (b) Quantitative analysis of confocal microscopy data. The mean fluorescence intensity, showing cell surface P2X7 receptor expression in peritoneal macrophages from uninfected mice (white bar), S. mansoni -infected (black bar), and TGF- β 1-treated (hatched bar; 5 ng/mL, for 24 h) mice, was obtained from pixels intensity using ImageJ software (see methods). Inset: negative controls (macrophages incubated with secondary antibodies only). n = 6 replicates from 3 animals for each condition (*** P < 0.001 versus control, P < 0.01 TGF- β 1 versus infected group; one-way ANOVA followed by post hoc Newman-Keuls test).

Article Snippet: Membranes were blocked for 1 h in Tris-buffered saline (TBS) with 2% nonfat milk and then incubated overnight with rabbit anti-P2X7 receptors (APR-004; Alomone Labs, Israel; 1 : 200; Peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acid residues 576–595 of P2X7 receptor intracellular, C-terminus) or mouse monoclonal anti- β -actin (1 : 20.000) primary antibodies.

Techniques: Confocal Microscopy, Expressing, Infection, Fluorescence, Software, Incubation

Journal: Mediators of Inflammation

Article Title: Macrophage P2X7 Receptor Function Is Reduced during Schistosomiasis: Putative Role of TGF- β 1

doi: 10.1155/2014/134974

Figure Lengend Snippet: Survival curve of C57BL/6 wild type or P2X7 KO infected with approximately 80 cercariae of S. mansoni . (P2X7 KO: n = 9 females and 7 males; C57BL/6 wild type: 4 females and 10 males). *** P < 0.0001 using the Mantel-Cox log-rank test.

Article Snippet: Membranes were blocked for 1 h in Tris-buffered saline (TBS) with 2% nonfat milk and then incubated overnight with rabbit anti-P2X7 receptors (APR-004; Alomone Labs, Israel; 1 : 200; Peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acid residues 576–595 of P2X7 receptor intracellular, C-terminus) or mouse monoclonal anti- β -actin (1 : 20.000) primary antibodies.

Techniques: Infection

Journal: Mediators of Inflammation

Article Title: Macrophage P2X7 Receptor Function Is Reduced during Schistosomiasis: Putative Role of TGF- β 1

doi: 10.1155/2014/134974

Figure Lengend Snippet: Intracellular Ca 2+ measurement (using Fura-2-AM) in macrophages subjected to treatment with the P2X7 agonist BzATP and/or with the antagonist A740003. (a) Changes in intracellular Ca 2+ levels in macrophages from control (black line) and S. mansoni -infected (red line) mice. Inset: representative images from macrophages (control) before (baseline) and after treatment with 100 μ M BzATP. (b) Changes in intracellular Ca 2+ levels ([Ca 2+ ] i ) in macrophages from control (white bars) or S. mansoni- infected (black bars) mice. Three plates were used for each condition/animal, 10 cells/plate were chosen randomly for imaging, and cells were obtained from three animals. Data were expressed as mean and SEM. * P < 0.05; *** P < 0.001 (one-way ANOVA followed by post hoc Newman-Keuls test).

Article Snippet: The following primary antibodies were used in this work: rabbit polyclonal anti-P2X7 receptors (APR-004 and APR-008; Alomone Labs, Israel); mouse monoclonal anti- β -actin (Santa Cruz Biotechnology, USA); rat anti-F4/80 (Biolegend, USA); rat anti-F4/80 FITC (AbD Serotec, USA).

Techniques: Infection, Imaging

Journal: Mediators of Inflammation

Article Title: Macrophage P2X7 Receptor Function Is Reduced during Schistosomiasis: Putative Role of TGF- β 1

doi: 10.1155/2014/134974

Figure Lengend Snippet: P2X7 receptors expression in peritoneal macrophages. (a) Macrophages from uninfected (white bar) and S. mansoni -infected (black bar) mice express similar levels of P2X7 protein. n = 9 replicates from three different animals, for each group. The images above are representative Western blots (U = uninfected and I = infected). (b) P2X7 receptors expression in peritoneal macrophages from control mice (white bar, untreated) or TGF- β 1-treated cells (5 ng/mL, 24 h; black bar) are also similar. The images above are representative Western blots (U = uninfected; TGF- β 1= TGF- β 1-treated macrophages). Lysates from P2X7 KO macrophages were used as negative controls for P2X7 protein expression. Cells lysates were obtained from three animals for each group. (c) Quantitative RT-PCR (qRT-PCR) analysis of P2X7 receptor mRNA levels in peritoneal macrophages. Similar levels of P2X7 mRNA were found in uninfected (white bar), S. mansoni -infected mice (black bar), and TGF- β 1-treated (5 ng/mL, 24 h; hatched bar) mouse macrophages. N = 3–5 samples per group (using different animals).

Article Snippet: The following primary antibodies were used in this work: rabbit polyclonal anti-P2X7 receptors (APR-004 and APR-008; Alomone Labs, Israel); mouse monoclonal anti- β -actin (Santa Cruz Biotechnology, USA); rat anti-F4/80 (Biolegend, USA); rat anti-F4/80 FITC (AbD Serotec, USA).

Techniques: Expressing, Infection, Western Blot, Quantitative RT-PCR

Journal: Mediators of Inflammation

Article Title: Macrophage P2X7 Receptor Function Is Reduced during Schistosomiasis: Putative Role of TGF- β 1

doi: 10.1155/2014/134974

Figure Lengend Snippet: Confocal microscopy analysis of P2X7 receptor expression on the cell surface of macrophages. Cells were labelled with antibodies recognizing P2X7 receptors (green) and F4/80+ (red) as well as with DAPI (blue). (a) Representative images showing macrophages from uninfected (left column), S. mansoni -infected (middle column), and TGF- β 1-treated (right column; 5 ng/mL, for 24 h) mice. The bottom row shows orthogonal slices of the representative labelled cells, with red arrows marking P2X7 expression on the plasma membrane. (b) Quantitative analysis of confocal microscopy data. The mean fluorescence intensity, showing cell surface P2X7 receptor expression in peritoneal macrophages from uninfected mice (white bar), S. mansoni -infected (black bar), and TGF- β 1-treated (hatched bar; 5 ng/mL, for 24 h) mice, was obtained from pixels intensity using ImageJ software (see methods). Inset: negative controls (macrophages incubated with secondary antibodies only). n = 6 replicates from 3 animals for each condition (*** P < 0.001 versus control, P < 0.01 TGF- β 1 versus infected group; one-way ANOVA followed by post hoc Newman-Keuls test).

Article Snippet: The following primary antibodies were used in this work: rabbit polyclonal anti-P2X7 receptors (APR-004 and APR-008; Alomone Labs, Israel); mouse monoclonal anti- β -actin (Santa Cruz Biotechnology, USA); rat anti-F4/80 (Biolegend, USA); rat anti-F4/80 FITC (AbD Serotec, USA).

Techniques: Confocal Microscopy, Expressing, Infection, Fluorescence, Software, Incubation

Journal: Mediators of Inflammation

Article Title: Macrophage P2X7 Receptor Function Is Reduced during Schistosomiasis: Putative Role of TGF- β 1

doi: 10.1155/2014/134974

Figure Lengend Snippet: Survival curve of C57BL/6 wild type or P2X7 KO infected with approximately 80 cercariae of S. mansoni . (P2X7 KO: n = 9 females and 7 males; C57BL/6 wild type: 4 females and 10 males). *** P < 0.0001 using the Mantel-Cox log-rank test.

Article Snippet: The following primary antibodies were used in this work: rabbit polyclonal anti-P2X7 receptors (APR-004 and APR-008; Alomone Labs, Israel); mouse monoclonal anti- β -actin (Santa Cruz Biotechnology, USA); rat anti-F4/80 (Biolegend, USA); rat anti-F4/80 FITC (AbD Serotec, USA).

Techniques: Infection

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: The sequences of oligonucleotide primers.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques:

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: The antibodies for immunoblotting.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Western Blot, Concentration Assay

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: The antibodies for fluorescence labeling.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Fluorescence, Labeling, Concentration Assay

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: Mechanical threshold and P2X7R and p-p38 expression. (a) Mechanical threshold after BTZ injection. (b, c) Western blot for P2X7R expression after BTZ treatment. (d) Immunofluorescence location of P2X7R in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. P2X7R is not expressed in NF-200-positive neurons. P2X7R is expressed in GFAP-labeled satellite glial cells (SGCs). (e) Immunofluorescence location of p-p38 in DRG. The arrows indicate the typical single- or double-labeled DRG neurons and satellite cells. p-p38 is expressed in both MAP2-labeled neurons and GFAP-labeled SGCs. (f) Immunofluorescence location of P2X7R in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. P2X7R is expressed mainly in Iba-1-labeled microglial cells rather than in GFAP-labeled astrocytes and MAP2-labeled neurons. (g) Immunofluorescence location of p-p38 in SDH. The arrows indicate the typical single-labeled and double-labeled cells in SDH. p-p38 is expressed mainly in Iba-1-labeled microglial cells. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Injection, Western Blot, Immunofluorescence, Labeling

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: p38 mRNA expression and p38 phosphorylation in DRG after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) p-p38 immunofluorescence labeling. The arrows show the typical p-p38 single-labeled DRG cells. (e) p-p38 fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition, Western Blot, Immunofluorescence, Labeling, Fluorescence

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: p38 mRNA expression and p38 phosphorylation in SDH after inhibition of P2X7R with BBG. (a) p38 mRNA levels. (b) p-p38 protein immunoblotting bands. (c) p-p38 protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: IL-1 β , IL-6, and TNF- α mRNA expression in DRG and SDH after inhibition of P2X7R. (a) DRG IL-1 β mRNA. (b) DRG IL-6 mRNA. (c) DRG TNF- α mRNA. (d) SDH IL-1 β mRNA. (e) SDH IL-6 mRNA. (f) SDH TNF- α mRNA. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: P2X7R mRNA and protein expression in DRG after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and GFAP coexpression fluorescence labeling for SGCs. The arrows indicate the typical single-labeled and double-labeled DRG satellite cells. (e) P2X7R and GFAP coexpression fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: P2X7R mRNA and protein expression in SDH after inhibition of p38 phosphorylation. (a) P2X7R mRNA levels. (b) P2X7R protein immunoblotting bands. (c) P2X7R protein levels. (d) P2X7R and p-p38 coexpression fluorescence labeling. The arrows indicate the typical single-labeled and double-labeled SDH microglia. (e) P2X7R and p-p38 colocalization fluorescence density. Scale bar = 50 μ m. Mean ± SEM ( n = 5). ∗ P < 0.05; ∗∗ P < 0.01; ∗∗∗ P < 0.001.

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Expressing, Inhibition, Western Blot, Fluorescence, Labeling

Journal: BioMed Research International

Article Title: The Actions and Mechanisms of P2X7R and p38 MAPK Activation in Mediating Bortezomib-Induced Neuropathic Pain

doi: 10.1155/2020/8143754

Figure Lengend Snippet: Mechanical threshold alterations after inhibition of P2X7R or p38. (a) Mechanical threshold after inhibition of P2X7R. (b) Mechanical threshold after inhibition of p38. Mean ± SEM ( n = 5). ∗∗∗ P < 0.001 (vs. control); # P < 0.05; ## P < 0.01 (vs. BTZ group).

Article Snippet: Primary , Rabbit anti-P2X7R monoclonal IgG , 1 : 400 , Alomone Labs, Jerusalem, Israel.

Techniques: Inhibition

Journal: BMC Neuroscience

Article Title: Hypoxia induced amoeboid microglial cell activation in postnatal rat brain is mediated by ATP receptor P2X4

doi: 10.1186/1471-2202-12-111

Figure Lengend Snippet: P2X4, P2X7 and P2Y12 immunoexpression in AMC in postnatal rat brain . By immunofluorescence labeling, P2X4 is colocalized with OX42 in the amoeboid microglial cells. Colocalization of OX42 and P2X4 in cells (arrows) is seen in the corpus callosum and subventricular zone (Aa-c, Scale bar = 100 μm). Immunopositive cells are round and have a typical morphology of amoeboid microglial cells (B/Ca-c. Scale bar = 20 μm). The amoeboid microglial cells exhibit a stronger P2X4 immunoreactivity compared with that of P2X7 and P2Y12 (D/Ea-c, Scale bar = 20 μm.).

Article Snippet: The sections were then incubated at 22-24°C with rabbit anti-P2X4 polyclonal primary antibody (1: 200, Alomone, Cat: APR-002, Yiselie), rabbit anti-P2X7 polyclonal primary antibody (1: 200, Alomone, Cat: APR-004, Yiselie), and rabbit anti-P2Y12 polyclonal primary antibody (1: 200, Alomone, Cat: APR-012, Yiselie) overnight.

Techniques: Immunofluorescence, Labeling