p2x7r  (Alomone Labs)


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    Structured Review

    Alomone Labs p2x7r
    Exposure to high extracellular glucose alters expression of purinergic receptors and pannexin 1 channels in MLO-Y4 osteocytes and MOB-C osteoblasts. Relative protein expression levels of P2R subtypes (P2Y 1 R, P2Y 2 R, P2Y 4 R, <t>P2X7R</t> and P2X3R) and Panx1 in MLO-Y4 (A) and MOB-C (B) cultured in control low glucose (LG), high glucose (HG) and mannitol media (Data are presented as the means ± SEM; ***P
    P2x7r, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 96 stars, based on 1 article reviews
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    p2x7r - by Bioz Stars, 2021-12
    96/100 stars

    Images

    1) Product Images from "P2X7R-Panx1 Complex Impairs Bone Mechanosignaling under High Glucose Levels Associated with Type-1 Diabetes"

    Article Title: P2X7R-Panx1 Complex Impairs Bone Mechanosignaling under High Glucose Levels Associated with Type-1 Diabetes

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0155107

    Exposure to high extracellular glucose alters expression of purinergic receptors and pannexin 1 channels in MLO-Y4 osteocytes and MOB-C osteoblasts. Relative protein expression levels of P2R subtypes (P2Y 1 R, P2Y 2 R, P2Y 4 R, P2X7R and P2X3R) and Panx1 in MLO-Y4 (A) and MOB-C (B) cultured in control low glucose (LG), high glucose (HG) and mannitol media (Data are presented as the means ± SEM; ***P
    Figure Legend Snippet: Exposure to high extracellular glucose alters expression of purinergic receptors and pannexin 1 channels in MLO-Y4 osteocytes and MOB-C osteoblasts. Relative protein expression levels of P2R subtypes (P2Y 1 R, P2Y 2 R, P2Y 4 R, P2X7R and P2X3R) and Panx1 in MLO-Y4 (A) and MOB-C (B) cultured in control low glucose (LG), high glucose (HG) and mannitol media (Data are presented as the means ± SEM; ***P

    Techniques Used: Expressing, Cell Culture

    P2X7R and pannexin 1 channels form molecular complexes in bone cells. P2X7R and Panx1 protein-protein interaction was assessed by immunoprecipitating lysates from long bone tissue, MLO-Y4 osteocytes and MOB-C osteoblasts with P2X7R antibody followed by Western blotting with Panx1 and P2X7R antibodies. “+” lanes represent samples with P2X7R antibody and “-” lanes represent samples with no antibody. Note: Both MLO-Y4 and MOB-C express two bands of P2X7R [functional glycosylated form at ~79kD and a splice variant at ~ 56kD [ 56 ]]. Bands corresponding to IgG heavy chain (~50 kD) are indicated on the blot with P2X7R antibody.
    Figure Legend Snippet: P2X7R and pannexin 1 channels form molecular complexes in bone cells. P2X7R and Panx1 protein-protein interaction was assessed by immunoprecipitating lysates from long bone tissue, MLO-Y4 osteocytes and MOB-C osteoblasts with P2X7R antibody followed by Western blotting with Panx1 and P2X7R antibodies. “+” lanes represent samples with P2X7R antibody and “-” lanes represent samples with no antibody. Note: Both MLO-Y4 and MOB-C express two bands of P2X7R [functional glycosylated form at ~79kD and a splice variant at ~ 56kD [ 56 ]]. Bands corresponding to IgG heavy chain (~50 kD) are indicated on the blot with P2X7R antibody.

    Techniques Used: Western Blot, Functional Assay, Variant Assay

    P2X7R and pannexin 1 channels contribute to OFSS-induced ATP release from MLO-Y4 cells. Comparison of OFSS-induced ATP release from MLO-Y4 cells under control glucose condition (Ctrl: 100 mg/dL of glucose) and in the presence of P2X7R blockers (10 μM A438079, 10 μM A740003) or Panx1 blocker (100 nM MFQ) or combined A438079 + MFQ or A740003 + MFQ. ATP release under each condition was normalized by number of counted cells (**** P
    Figure Legend Snippet: P2X7R and pannexin 1 channels contribute to OFSS-induced ATP release from MLO-Y4 cells. Comparison of OFSS-induced ATP release from MLO-Y4 cells under control glucose condition (Ctrl: 100 mg/dL of glucose) and in the presence of P2X7R blockers (10 μM A438079, 10 μM A740003) or Panx1 blocker (100 nM MFQ) or combined A438079 + MFQ or A740003 + MFQ. ATP release under each condition was normalized by number of counted cells (**** P

    Techniques Used:

    Exposure to high glucose alters the amplitudes of Ca 2+ responses induced in (A) MLO-Y4 by P2X7R stimulation and in (B) MOB-C by P2Y 2 R/P2Y 4 R stimulation. Dose-response curves obtained for the P2X7R agonist BzATP (0.1–1000μM) in MLO-Y4 cells grown in low glucose (LG) and in high glucose (HG) (A) and for the P2Y2/P2Y4R agonist UTP (1–100μM) in MOB-C cells grown in LG and HG (B) conditions. Each point in the dose-response curve corresponds to average amplitude of Ca 2+ responses normalized to control LG maximal amplitude. Data are presented as the means ± SEM, n = 6 independent experiments per condition. Total number of cells analyzed: (A) 431 under HG and 457 under LG; (B) 549 under HG and 620 under LG. Student t-test: *** P
    Figure Legend Snippet: Exposure to high glucose alters the amplitudes of Ca 2+ responses induced in (A) MLO-Y4 by P2X7R stimulation and in (B) MOB-C by P2Y 2 R/P2Y 4 R stimulation. Dose-response curves obtained for the P2X7R agonist BzATP (0.1–1000μM) in MLO-Y4 cells grown in low glucose (LG) and in high glucose (HG) (A) and for the P2Y2/P2Y4R agonist UTP (1–100μM) in MOB-C cells grown in LG and HG (B) conditions. Each point in the dose-response curve corresponds to average amplitude of Ca 2+ responses normalized to control LG maximal amplitude. Data are presented as the means ± SEM, n = 6 independent experiments per condition. Total number of cells analyzed: (A) 431 under HG and 457 under LG; (B) 549 under HG and 620 under LG. Student t-test: *** P

    Techniques Used:

    2) Product Images from "Loss of P2X7 Receptor Plasma Membrane Expression and Function in Pathogenic B220+ Double-Negative T Lymphocytes of Autoimmune MRL/lpr Mice"

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

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0052161

    Expression of P2X7R and CD39 in T-cell subsets. (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 [22] . 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 [34] 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.
    Figure Legend Snippet: Expression of P2X7R and CD39 in T-cell subsets. (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 [22] . 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 [34] 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.

    Techniques Used: Expressing, Mouse Assay, Flow Cytometry, Cytometry, FACS, Purification, Quantitative RT-PCR, Polymerase Chain Reaction, Amplification, Western Blot, Affinity Purification, Staining, Generated

    Dose–response experiments for ATP-induced CD62L shedding on T lymphocytes. 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.
    Figure Legend Snippet: Dose–response experiments for ATP-induced CD62L shedding on T lymphocytes. 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.

    Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry, Expressing

    P2X7R antagonist KN-62 inhibits ATP-induced CD62L shedding and pore formation in MRL +/+ T lymphocytes. Spleen cells from 3 to 4-mo-old MRL +/+ mice (n = 4 mice/group) were preincubated for 15 min at 37°C with or without 0.5, 1 and 5 µM KN-62 and then stimulated with 500 µM ATP for 30 min. Cells were then double-stained with anti-CD90 mAb and either anti-CD62L or YO-PRO-1 to assess by flow cytometry: (A) CD62L shedding or (B) pore formation in the gated CD90 + T-cell population stimulated (▪) or not (□) with ATP. Asterisks denote statistically significant differences between ATP-stimulated and unstimulated groups: * p ≤0.05; ** p ≤0.01; *** p ≤0.001.
    Figure Legend Snippet: P2X7R antagonist KN-62 inhibits ATP-induced CD62L shedding and pore formation in MRL +/+ T lymphocytes. Spleen cells from 3 to 4-mo-old MRL +/+ mice (n = 4 mice/group) were preincubated for 15 min at 37°C with or without 0.5, 1 and 5 µM KN-62 and then stimulated with 500 µM ATP for 30 min. Cells were then double-stained with anti-CD90 mAb and either anti-CD62L or YO-PRO-1 to assess by flow cytometry: (A) CD62L shedding or (B) pore formation in the gated CD90 + T-cell population stimulated (▪) or not (□) with ATP. Asterisks denote statistically significant differences between ATP-stimulated and unstimulated groups: * p ≤0.05; ** p ≤0.01; *** p ≤0.001.

    Techniques Used: Mouse Assay, Staining, Flow Cytometry, Cytometry

    P2X7R activity in T lymphocytes from MRL/lpr mice at early and late stages of the disease, and in B220 + and B220 – T lymphocytes from MRL +/+ and MRL/lpr mice. (A and B) Spleen cells from 4-mo-old MRL +/+ and 2- and 4-mo-old MRL/ lpr mice (n = 3 mice/group) were either left unstimulated (□) or stimulated with 500 µM ATP (▪) or 50 µM NAD (▪) for 45 min at 37°C. Cells were then double-stained with anti-CD90 mAb and either Annexin V or YO-PRO-1 to assess PS exposure and pore formation, respectively, in the gated CD90 + T-cell population by flow cytometry. (C–E) Spleen cells from 3 to 5-mo-old MRL +/+ and MRL /lpr (n = 5 mice/group) were either left unstimulated (□) or stimulated with 500 µM ATP (▪) or 50 µM NAD (▪) for 45 min at 37°C. Cells were then triple-stained with anti-CD90, anti-B220 and either anti-CD62L mAb, YO-PRO-1 or 7-AAD to assess CD62L shedding (C), pore formation (D) or cell death (E) in B220 – CD90 + and B220 + CD90 + T-cell subsets. Asterisks denote statistically significant differences between ATP- or NAD-stimulated and unstimulated groups: ** p ≤0.01; *** p ≤0.001.
    Figure Legend Snippet: P2X7R activity in T lymphocytes from MRL/lpr mice at early and late stages of the disease, and in B220 + and B220 – T lymphocytes from MRL +/+ and MRL/lpr mice. (A and B) Spleen cells from 4-mo-old MRL +/+ and 2- and 4-mo-old MRL/ lpr mice (n = 3 mice/group) were either left unstimulated (□) or stimulated with 500 µM ATP (▪) or 50 µM NAD (▪) for 45 min at 37°C. Cells were then double-stained with anti-CD90 mAb and either Annexin V or YO-PRO-1 to assess PS exposure and pore formation, respectively, in the gated CD90 + T-cell population by flow cytometry. (C–E) Spleen cells from 3 to 5-mo-old MRL +/+ and MRL /lpr (n = 5 mice/group) were either left unstimulated (□) or stimulated with 500 µM ATP (▪) or 50 µM NAD (▪) for 45 min at 37°C. Cells were then triple-stained with anti-CD90, anti-B220 and either anti-CD62L mAb, YO-PRO-1 or 7-AAD to assess CD62L shedding (C), pore formation (D) or cell death (E) in B220 – CD90 + and B220 + CD90 + T-cell subsets. Asterisks denote statistically significant differences between ATP- or NAD-stimulated and unstimulated groups: ** p ≤0.01; *** p ≤0.001.

    Techniques Used: Activity Assay, Mouse Assay, Staining, Flow Cytometry, Cytometry

    P2X7R activity in T lymphocytes from normal MRL +/+ and autoimmune MRL/lpr mice. Spleen cells from 3 to 4-mo-old MRL +/+ (□) and MRL/ lpr (▪) mice (n = 5 mice/group) were incubated with or without 500 µM ATP for 45 min (A–C) or 4 h (D) at 37°C. Cells were then double-stained with anti-CD90 mAb and either anti-CD62L mAb, YO-PRO-1, Annexin V or 7-AAD to assess in T cells by flow cytometry: (A) CD62L shedding; (B) pore formation; (C) cell surface PS exposure; (D) cell death. Numbers reported in the dot plots indicate the percentages of CD62L + CD90 + or YO-PRO-1 + CD90 + cells in the gated CD90 + T-cell population. Histograms correspond to the mean percentages ± SE (n = 5 mice/group) of double-stained cells in the gated CD90 + T-cell population. Asterisks denote statistically significant differences (*** p ≤0.001) between ATP-stimulated and unstimulated groups.
    Figure Legend Snippet: P2X7R activity in T lymphocytes from normal MRL +/+ and autoimmune MRL/lpr mice. Spleen cells from 3 to 4-mo-old MRL +/+ (□) and MRL/ lpr (▪) mice (n = 5 mice/group) were incubated with or without 500 µM ATP for 45 min (A–C) or 4 h (D) at 37°C. Cells were then double-stained with anti-CD90 mAb and either anti-CD62L mAb, YO-PRO-1, Annexin V or 7-AAD to assess in T cells by flow cytometry: (A) CD62L shedding; (B) pore formation; (C) cell surface PS exposure; (D) cell death. Numbers reported in the dot plots indicate the percentages of CD62L + CD90 + or YO-PRO-1 + CD90 + cells in the gated CD90 + T-cell population. Histograms correspond to the mean percentages ± SE (n = 5 mice/group) of double-stained cells in the gated CD90 + T-cell population. Asterisks denote statistically significant differences (*** p ≤0.001) between ATP-stimulated and unstimulated groups.

    Techniques Used: Activity Assay, Mouse Assay, Incubation, Staining, Flow Cytometry, Cytometry

    3) Product Images from "Genetically dissecting P2rx7 expression within the central nervous system using conditional humanized mice"

    Article Title: Genetically dissecting P2rx7 expression within the central nervous system using conditional humanized mice

    Journal: Purinergic Signalling

    doi: 10.1007/s11302-016-9546-z

    Analysis of P2X7R expression in the mouse brain by in situ hybridization using human- and mouse-specific riboprobes on conditional knockout mice. Depicted are coronal brain sections (overview, left columns ) and magnifications (hippocampus or cerebellum, right columns ) of control mice (−Cre) and respective conditional knockout mice generated by breeding to a Deleter-Cre , b Nes-Cre , c Nex-Cre , and d, e En1-cre mice (+Cre). White arrows indicate in situ hybridization signals in the white matter of the corpus callosum ( a ) and cerebellum ( e )
    Figure Legend Snippet: Analysis of P2X7R expression in the mouse brain by in situ hybridization using human- and mouse-specific riboprobes on conditional knockout mice. Depicted are coronal brain sections (overview, left columns ) and magnifications (hippocampus or cerebellum, right columns ) of control mice (−Cre) and respective conditional knockout mice generated by breeding to a Deleter-Cre , b Nes-Cre , c Nex-Cre , and d, e En1-cre mice (+Cre). White arrows indicate in situ hybridization signals in the white matter of the corpus callosum ( a ) and cerebellum ( e )

    Techniques Used: Expressing, In Situ Hybridization, Knock-Out, Mouse Assay, Generated

    Analysis of P2X7R expression in the mouse brain by RT-qPCR using conditional knockout mice. Relative expression of hP2RX7 was determined by real-time RT-qPCR using mRNA prepared from cortex, hippocampus, and cerebellum of heterozygous humanized mice ( P2rx7 +/hP2RX7 ; +/hP2RX7). Expression of the floxed human P2RX7 transcript was normalized to the expression of the endogenous murine P2rx7 transcript (≙100 %). Heterozygous knockout mice demonstrated the specificity of the RT-qPCR. Cortex, hippocampus, and cerebellum of heterozygous mice positive for brain region- or cell type-specifically expressed Cre recombinase were analyzed accordingly. t test, * p
    Figure Legend Snippet: Analysis of P2X7R expression in the mouse brain by RT-qPCR using conditional knockout mice. Relative expression of hP2RX7 was determined by real-time RT-qPCR using mRNA prepared from cortex, hippocampus, and cerebellum of heterozygous humanized mice ( P2rx7 +/hP2RX7 ; +/hP2RX7). Expression of the floxed human P2RX7 transcript was normalized to the expression of the endogenous murine P2rx7 transcript (≙100 %). Heterozygous knockout mice demonstrated the specificity of the RT-qPCR. Cortex, hippocampus, and cerebellum of heterozygous mice positive for brain region- or cell type-specifically expressed Cre recombinase were analyzed accordingly. t test, * p

    Techniques Used: Expressing, Quantitative RT-PCR, Knock-Out, Mouse Assay

    Comparison of BzATP-induced Yo-Pro-1 uptake of humanized and murine P2X7R expressing peritoneal macrophages. a Pore formation capacity of peritoneal macrophages expressing the murine and the humanized P2X7R. BzATP concentration-response curves for the mouse and humanized P2X7R following different incubation times. The humanized receptor shows higher sensitivity toward the agonist compared to the murine counterpart as determined by increasing concentrations of BzATP. b Peritoneal macrophages expressing the murine or humanized P2X7R show differential modulation of Yo-Pro-1 uptake by TFP upon concurrent stimulation with BzATP. Two-way RM-ANOVA + Bonferroni post hoc test, * p
    Figure Legend Snippet: Comparison of BzATP-induced Yo-Pro-1 uptake of humanized and murine P2X7R expressing peritoneal macrophages. a Pore formation capacity of peritoneal macrophages expressing the murine and the humanized P2X7R. BzATP concentration-response curves for the mouse and humanized P2X7R following different incubation times. The humanized receptor shows higher sensitivity toward the agonist compared to the murine counterpart as determined by increasing concentrations of BzATP. b Peritoneal macrophages expressing the murine or humanized P2X7R show differential modulation of Yo-Pro-1 uptake by TFP upon concurrent stimulation with BzATP. Two-way RM-ANOVA + Bonferroni post hoc test, * p

    Techniques Used: Expressing, Concentration Assay, Incubation

    Assessment of P2rx7 splice variants in P2X7R knockout mice. a Depicted are the 5 described P2rx7 splice variants. Exons differing from the most abundant variant P2rx7-a are highlighted in blue . Translation start and stop sites are indicated by asterisks . Exon sizes are true to scale. Primers used for nested RT-PCR are schematically depicted above and below each transcript and are indicated by capital letters and arrows . b P2X7R knockout (KO) mice lack all P2rx7 splice variants as determined by PCR. The remaining amplicons lack exon 2 and therefore do not represent transcripts translated to functional proteins
    Figure Legend Snippet: Assessment of P2rx7 splice variants in P2X7R knockout mice. a Depicted are the 5 described P2rx7 splice variants. Exons differing from the most abundant variant P2rx7-a are highlighted in blue . Translation start and stop sites are indicated by asterisks . Exon sizes are true to scale. Primers used for nested RT-PCR are schematically depicted above and below each transcript and are indicated by capital letters and arrows . b P2X7R knockout (KO) mice lack all P2rx7 splice variants as determined by PCR. The remaining amplicons lack exon 2 and therefore do not represent transcripts translated to functional proteins

    Techniques Used: Knock-Out, Mouse Assay, Variant Assay, Reverse Transcription Polymerase Chain Reaction, Polymerase Chain Reaction, Functional Assay

    Generation and validation of P2X7R knockout mice. a The floxed humanized allele allows for Cre recombinase-mediated excision of the human P2RX7 resulting in a knockout (KO) allele. b Genotyping by PCR discriminates between humanized (613 bp) and KO allele (222 bp). c Representative Western blot demonstrating the loss of P2X7R protein in peripheral tissues of KO mice (SG, salivary gland; Spl, spleen). d Assessment of Yo-Pro-1 uptake shows that peritoneal macrophages obtained from KO mice lost their pore formation capacity compared to mouse or humanized cells upon prolonged stimulation of the receptor with 50 μM BzATP (two-way RM-ANOVA + Bonferroni post hoc test, * p
    Figure Legend Snippet: Generation and validation of P2X7R knockout mice. a The floxed humanized allele allows for Cre recombinase-mediated excision of the human P2RX7 resulting in a knockout (KO) allele. b Genotyping by PCR discriminates between humanized (613 bp) and KO allele (222 bp). c Representative Western blot demonstrating the loss of P2X7R protein in peripheral tissues of KO mice (SG, salivary gland; Spl, spleen). d Assessment of Yo-Pro-1 uptake shows that peritoneal macrophages obtained from KO mice lost their pore formation capacity compared to mouse or humanized cells upon prolonged stimulation of the receptor with 50 μM BzATP (two-way RM-ANOVA + Bonferroni post hoc test, * p

    Techniques Used: Knock-Out, Mouse Assay, Polymerase Chain Reaction, Western Blot

    4) Product Images from "The Purinergic Receptor P2X7 Triggers ?-Secretase-dependent Processing of the Amyloid Precursor Protein *"

    Article Title: The Purinergic Receptor P2X7 Triggers ?-Secretase-dependent Processing of the Amyloid Precursor Protein *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M110.200618

    Activation of the P2X7R induces APP cleavage by a metalloprotease. Neuro2a-hAPP cells preincubated for 1 h at 37 °C with 50 μ m Z-VAD-FMK ( A and B ) or increasing concentrations of GM6001 ( C and D ) or TAPI-2 ( E and F ), and stimulated with
    Figure Legend Snippet: Activation of the P2X7R induces APP cleavage by a metalloprotease. Neuro2a-hAPP cells preincubated for 1 h at 37 °C with 50 μ m Z-VAD-FMK ( A and B ) or increasing concentrations of GM6001 ( C and D ) or TAPI-2 ( E and F ), and stimulated with

    Techniques Used: Activation Assay

    P2X7R-dependent extracellular Ca 2+ influx is not involved in sAPP release. A , Neuro2a-hAPP cells were loaded with 2 μ m Fura-2 AM for 30 min. Then, these cells were stimulated with 300 μ m Bz-ATP (■) or 1 μ m thapsigargin
    Figure Legend Snippet: P2X7R-dependent extracellular Ca 2+ influx is not involved in sAPP release. A , Neuro2a-hAPP cells were loaded with 2 μ m Fura-2 AM for 30 min. Then, these cells were stimulated with 300 μ m Bz-ATP (■) or 1 μ m thapsigargin

    Techniques Used:

    Role of MAP kinase pathways in P2X7R-dependent α-cleavage of APP. A , a kinetic study of Erk1/2 phosphorylation after 1 m m Bz-ATP stimulation is shown in the upper left Western blot. The effect of MEK inhibitor is shown on the upper right and
    Figure Legend Snippet: Role of MAP kinase pathways in P2X7R-dependent α-cleavage of APP. A , a kinetic study of Erk1/2 phosphorylation after 1 m m Bz-ATP stimulation is shown in the upper left Western blot. The effect of MEK inhibitor is shown on the upper right and

    Techniques Used: Western Blot

    P2X7R-mediated APP processing is independent of ADAM9, ADAM10, and ADAM17. A , cell lysates of Neuro2a-hAPP cells transfected with control, ADAM10 or ADAM17 siRNA were analyzed by Western blot using anti-ADAM10 or anti-ADAM17 Abs. B , Neuro2a-hAPP cells
    Figure Legend Snippet: P2X7R-mediated APP processing is independent of ADAM9, ADAM10, and ADAM17. A , cell lysates of Neuro2a-hAPP cells transfected with control, ADAM10 or ADAM17 siRNA were analyzed by Western blot using anti-ADAM10 or anti-ADAM17 Abs. B , Neuro2a-hAPP cells

    Techniques Used: Transfection, Western Blot

    Bz-ATP stimulation induces sAPP release via activation of P2X7R. Neuro2a-hAPP cells were preincubated at 37 °C for 1 h with 10 μ m and 50 μ m BBG ( A ), for 2 h with 300 μ m oATP ( B ), and for 30 min with 10 μ m A438079
    Figure Legend Snippet: Bz-ATP stimulation induces sAPP release via activation of P2X7R. Neuro2a-hAPP cells were preincubated at 37 °C for 1 h with 10 μ m and 50 μ m BBG ( A ), for 2 h with 300 μ m oATP ( B ), and for 30 min with 10 μ m A438079

    Techniques Used: Activation Assay

    5) Product Images from "Atheroprone flow activates inflammation via endothelial ATP-dependent P2X7-p38 signalling"

    Article Title: Atheroprone flow activates inflammation via endothelial ATP-dependent P2X7-p38 signalling

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvx213

    P2X7 receptor activity occurs exclusively in HUVEC under atheroprone flow. Representative single cell ATP-induced calcium responses in atheroprotected ( A ) or atheroprone ( B, C ) flow conditioned HUVEC after thapsigargin pre-treatment ± the P2X7 inhibitor 10 µM A438079 (P2X7i) or siRNA control and P2X7 knockdown ( C ) and analysed by measuring the average area under the curve ( n = 4, 175 cells per donor, * indicates P ≤ 0.05 using a paired t -test). Values are mean ± S.E.M.
    Figure Legend Snippet: P2X7 receptor activity occurs exclusively in HUVEC under atheroprone flow. Representative single cell ATP-induced calcium responses in atheroprotected ( A ) or atheroprone ( B, C ) flow conditioned HUVEC after thapsigargin pre-treatment ± the P2X7 inhibitor 10 µM A438079 (P2X7i) or siRNA control and P2X7 knockdown ( C ) and analysed by measuring the average area under the curve ( n = 4, 175 cells per donor, * indicates P ≤ 0.05 using a paired t -test). Values are mean ± S.E.M.

    Techniques Used: Activity Assay

    Expression of ATP-gated P2X7 receptors is enhanced under atheroprone flow. ( A ) Western blot and densitometry of P2X7 in flow conditioned HUVEC using the ibidi flow system ( n = 6, ** indicates P ≤ 0.01 using a paired t -test). P2X7 is shown by an arrow, validated by P2X7 knockdown, shown in Supplementary material online , Figure 4 B. ( B ) Western blot and densitometry of P2X7 in flow conditioned HUVEC using the orbital shaker system ( n = 4, ** indicates P ≤ 0.01 using a paired t -test). ( C ) Representative en face immunostaining for P2X7 (red) on wildtype C57BL/6 mice at atheroprotective (outer curvature) and atheroprone (inner curvature) regions of the aorta (scale = 25 µm). Endothelial cells were identified by staining with CD31 (green) and cell nuclei were stained using TO-PRO (blue). P2X7 expression was analysed by measuring the relative fluorescent intensity at sites protected or prone to atherosclerosis. Surface expression was measured by measuring P2X7 fluorescence at sites co-localized with the endothelial cell surface marker CD31. Relative fluorescent intensities for P2X7 were corrected against the relative fluorescent intensity of IgG performed on the descending aorta ( n = 5, * indicates P ≤ 0.05 and *** indicates P ≤ 0.001 using a paired t -test). Values are mean ± S.E.M.
    Figure Legend Snippet: Expression of ATP-gated P2X7 receptors is enhanced under atheroprone flow. ( A ) Western blot and densitometry of P2X7 in flow conditioned HUVEC using the ibidi flow system ( n = 6, ** indicates P ≤ 0.01 using a paired t -test). P2X7 is shown by an arrow, validated by P2X7 knockdown, shown in Supplementary material online , Figure 4 B. ( B ) Western blot and densitometry of P2X7 in flow conditioned HUVEC using the orbital shaker system ( n = 4, ** indicates P ≤ 0.01 using a paired t -test). ( C ) Representative en face immunostaining for P2X7 (red) on wildtype C57BL/6 mice at atheroprotective (outer curvature) and atheroprone (inner curvature) regions of the aorta (scale = 25 µm). Endothelial cells were identified by staining with CD31 (green) and cell nuclei were stained using TO-PRO (blue). P2X7 expression was analysed by measuring the relative fluorescent intensity at sites protected or prone to atherosclerosis. Surface expression was measured by measuring P2X7 fluorescence at sites co-localized with the endothelial cell surface marker CD31. Relative fluorescent intensities for P2X7 were corrected against the relative fluorescent intensity of IgG performed on the descending aorta ( n = 5, * indicates P ≤ 0.05 and *** indicates P ≤ 0.001 using a paired t -test). Values are mean ± S.E.M.

    Techniques Used: Expressing, Western Blot, Immunostaining, Mouse Assay, Staining, Fluorescence, Marker

    P2X7 regulates E-selectin expression at sites susceptible to atherosclerosis in vivo . Representative en face immunostaining for E-selectin (red) on wildtype or P2X7 −/− BALB/c mice at atheroprotective (outer curvature) and atheroprone (inner curvature) sites of the aorta ( n = 4–5) (scale = 25 µm). Endothelial cells were identified by staining with CD31 (green) and cell nuclei were stained using TO-PRO (blue). E-selectin expression was analysed by measuring the relative fluorescent intensity at sites of protected or prone to atherosclerosis ( n = 4–5, * indicates P ≤ 0.05, ** indicates P ≤ 0.01 and *** indicates P ≤ 0.001 using a two-way ANOVA). Relative fluorescent intensities for P2X7 were corrected against the relative fluorescent intensity of IgG. Values are mean ± S.E.M.
    Figure Legend Snippet: P2X7 regulates E-selectin expression at sites susceptible to atherosclerosis in vivo . Representative en face immunostaining for E-selectin (red) on wildtype or P2X7 −/− BALB/c mice at atheroprotective (outer curvature) and atheroprone (inner curvature) sites of the aorta ( n = 4–5) (scale = 25 µm). Endothelial cells were identified by staining with CD31 (green) and cell nuclei were stained using TO-PRO (blue). E-selectin expression was analysed by measuring the relative fluorescent intensity at sites of protected or prone to atherosclerosis ( n = 4–5, * indicates P ≤ 0.05, ** indicates P ≤ 0.01 and *** indicates P ≤ 0.001 using a two-way ANOVA). Relative fluorescent intensities for P2X7 were corrected against the relative fluorescent intensity of IgG. Values are mean ± S.E.M.

    Techniques Used: Expressing, In Vivo, Immunostaining, Mouse Assay, Staining

    Atheroprone flow mediated inflammatory signalling is regulated by P2X7 responses. qPCR analysis of E-selectin ( A ) and IL-8 ( B ) in HUVEC conditioned with atheroprotective or atheroprone flow using the ibidi system ± the P2X7 inhibitor 10 µM A438079 (P2X7i) ( n = 5, * indicates P ≤ 0.05 using a two-way ANOVA). ( C ) IL-8 release measured by ELISA in the supernatant of HUVEC conditioned under atheroprotective or atheroprone flow using the ibidi system ± the P2X7 inhibitor 10 µM A438079 (P2X7i) ( n = 9, * indicates P ≤ 0.05, *** indicates p =
    Figure Legend Snippet: Atheroprone flow mediated inflammatory signalling is regulated by P2X7 responses. qPCR analysis of E-selectin ( A ) and IL-8 ( B ) in HUVEC conditioned with atheroprotective or atheroprone flow using the ibidi system ± the P2X7 inhibitor 10 µM A438079 (P2X7i) ( n = 5, * indicates P ≤ 0.05 using a two-way ANOVA). ( C ) IL-8 release measured by ELISA in the supernatant of HUVEC conditioned under atheroprotective or atheroprone flow using the ibidi system ± the P2X7 inhibitor 10 µM A438079 (P2X7i) ( n = 9, * indicates P ≤ 0.05, *** indicates p =

    Techniques Used: Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

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    Alomone Labs p2x7r
    Exposure to high extracellular glucose alters expression of purinergic receptors and pannexin 1 channels in MLO-Y4 osteocytes and MOB-C osteoblasts. Relative protein expression levels of P2R subtypes (P2Y 1 R, P2Y 2 R, P2Y 4 R, <t>P2X7R</t> and P2X3R) and Panx1 in MLO-Y4 (A) and MOB-C (B) cultured in control low glucose (LG), high glucose (HG) and mannitol media (Data are presented as the means ± SEM; ***P
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    Exposure to high extracellular glucose alters expression of purinergic receptors and pannexin 1 channels in MLO-Y4 osteocytes and MOB-C osteoblasts. Relative protein expression levels of P2R subtypes (P2Y 1 R, P2Y 2 R, P2Y 4 R, P2X7R and P2X3R) and Panx1 in MLO-Y4 (A) and MOB-C (B) cultured in control low glucose (LG), high glucose (HG) and mannitol media (Data are presented as the means ± SEM; ***P

    Journal: PLoS ONE

    Article Title: P2X7R-Panx1 Complex Impairs Bone Mechanosignaling under High Glucose Levels Associated with Type-1 Diabetes

    doi: 10.1371/journal.pone.0155107

    Figure Lengend Snippet: Exposure to high extracellular glucose alters expression of purinergic receptors and pannexin 1 channels in MLO-Y4 osteocytes and MOB-C osteoblasts. Relative protein expression levels of P2R subtypes (P2Y 1 R, P2Y 2 R, P2Y 4 R, P2X7R and P2X3R) and Panx1 in MLO-Y4 (A) and MOB-C (B) cultured in control low glucose (LG), high glucose (HG) and mannitol media (Data are presented as the means ± SEM; ***P

    Article Snippet: Western blotting of the immunoprecipitated complexes was performed and membranes were probed with antibodies against Panx1 and P2X7R.

    Techniques: Expressing, Cell Culture

    P2X7R and pannexin 1 channels form molecular complexes in bone cells. P2X7R and Panx1 protein-protein interaction was assessed by immunoprecipitating lysates from long bone tissue, MLO-Y4 osteocytes and MOB-C osteoblasts with P2X7R antibody followed by Western blotting with Panx1 and P2X7R antibodies. “+” lanes represent samples with P2X7R antibody and “-” lanes represent samples with no antibody. Note: Both MLO-Y4 and MOB-C express two bands of P2X7R [functional glycosylated form at ~79kD and a splice variant at ~ 56kD [ 56 ]]. Bands corresponding to IgG heavy chain (~50 kD) are indicated on the blot with P2X7R antibody.

    Journal: PLoS ONE

    Article Title: P2X7R-Panx1 Complex Impairs Bone Mechanosignaling under High Glucose Levels Associated with Type-1 Diabetes

    doi: 10.1371/journal.pone.0155107

    Figure Lengend Snippet: P2X7R and pannexin 1 channels form molecular complexes in bone cells. P2X7R and Panx1 protein-protein interaction was assessed by immunoprecipitating lysates from long bone tissue, MLO-Y4 osteocytes and MOB-C osteoblasts with P2X7R antibody followed by Western blotting with Panx1 and P2X7R antibodies. “+” lanes represent samples with P2X7R antibody and “-” lanes represent samples with no antibody. Note: Both MLO-Y4 and MOB-C express two bands of P2X7R [functional glycosylated form at ~79kD and a splice variant at ~ 56kD [ 56 ]]. Bands corresponding to IgG heavy chain (~50 kD) are indicated on the blot with P2X7R antibody.

    Article Snippet: Western blotting of the immunoprecipitated complexes was performed and membranes were probed with antibodies against Panx1 and P2X7R.

    Techniques: Western Blot, Functional Assay, Variant Assay

    P2X7R and pannexin 1 channels contribute to OFSS-induced ATP release from MLO-Y4 cells. Comparison of OFSS-induced ATP release from MLO-Y4 cells under control glucose condition (Ctrl: 100 mg/dL of glucose) and in the presence of P2X7R blockers (10 μM A438079, 10 μM A740003) or Panx1 blocker (100 nM MFQ) or combined A438079 + MFQ or A740003 + MFQ. ATP release under each condition was normalized by number of counted cells (**** P

    Journal: PLoS ONE

    Article Title: P2X7R-Panx1 Complex Impairs Bone Mechanosignaling under High Glucose Levels Associated with Type-1 Diabetes

    doi: 10.1371/journal.pone.0155107

    Figure Lengend Snippet: P2X7R and pannexin 1 channels contribute to OFSS-induced ATP release from MLO-Y4 cells. Comparison of OFSS-induced ATP release from MLO-Y4 cells under control glucose condition (Ctrl: 100 mg/dL of glucose) and in the presence of P2X7R blockers (10 μM A438079, 10 μM A740003) or Panx1 blocker (100 nM MFQ) or combined A438079 + MFQ or A740003 + MFQ. ATP release under each condition was normalized by number of counted cells (**** P

    Article Snippet: Western blotting of the immunoprecipitated complexes was performed and membranes were probed with antibodies against Panx1 and P2X7R.

    Techniques:

    Exposure to high glucose alters the amplitudes of Ca 2+ responses induced in (A) MLO-Y4 by P2X7R stimulation and in (B) MOB-C by P2Y 2 R/P2Y 4 R stimulation. Dose-response curves obtained for the P2X7R agonist BzATP (0.1–1000μM) in MLO-Y4 cells grown in low glucose (LG) and in high glucose (HG) (A) and for the P2Y2/P2Y4R agonist UTP (1–100μM) in MOB-C cells grown in LG and HG (B) conditions. Each point in the dose-response curve corresponds to average amplitude of Ca 2+ responses normalized to control LG maximal amplitude. Data are presented as the means ± SEM, n = 6 independent experiments per condition. Total number of cells analyzed: (A) 431 under HG and 457 under LG; (B) 549 under HG and 620 under LG. Student t-test: *** P

    Journal: PLoS ONE

    Article Title: P2X7R-Panx1 Complex Impairs Bone Mechanosignaling under High Glucose Levels Associated with Type-1 Diabetes

    doi: 10.1371/journal.pone.0155107

    Figure Lengend Snippet: Exposure to high glucose alters the amplitudes of Ca 2+ responses induced in (A) MLO-Y4 by P2X7R stimulation and in (B) MOB-C by P2Y 2 R/P2Y 4 R stimulation. Dose-response curves obtained for the P2X7R agonist BzATP (0.1–1000μM) in MLO-Y4 cells grown in low glucose (LG) and in high glucose (HG) (A) and for the P2Y2/P2Y4R agonist UTP (1–100μM) in MOB-C cells grown in LG and HG (B) conditions. Each point in the dose-response curve corresponds to average amplitude of Ca 2+ responses normalized to control LG maximal amplitude. Data are presented as the means ± SEM, n = 6 independent experiments per condition. Total number of cells analyzed: (A) 431 under HG and 457 under LG; (B) 549 under HG and 620 under LG. Student t-test: *** P

    Article Snippet: Western blotting of the immunoprecipitated complexes was performed and membranes were probed with antibodies against Panx1 and P2X7R.

    Techniques: