p2x7r (Alomone Labs)

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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

P2x7r, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Average 95 stars, based on 4 article reviews
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p2x7r - by Bioz Stars, 2022-08

95/100 stars

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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

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

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Alomone Labs rabbit polyclonal p2x7 c terminus antibody

The N terminus is important for the sensitivity of P2X7 receptor activity to cholesterol depletion. A , amino acid sequence alignment of the N-terminal region of human P2X1, P2X2, and P2X7 receptors, with conserved amino acids highlighted in gray , P2X1 aa20–23 and equivalent residues in P2X2 underlined , and the P2X7 residues Lys 17 and Val 18 in red. B , time course of dye uptake mediated by the K17R/V18M mutant and wild type P2X7, alone, pretreated with MCD and loaded with cholesterol ( chol ), normalized to the untreated WT P2X7. C , bar graph of the relative rate of dye uptake normalized to the untreated WT P2X7. Dye uptake mediated by the K17R/V18M mutant is not potentiated by MCD, but is inhibited by cholesterol loading (WT: n = 14 alone, n = 11 MCD, n = 3 cholesterol; mutants: n = 12 alone, n = 11 MCD, n = 3 cholesterol; *, p

Rabbit Polyclonal P2x7 C Terminus Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/rabbit polyclonal p2x7 c terminus antibody/product/Alomone Labs
Average 86 stars, based on 1 article reviews
Price from $9.99 to $1999.99

rabbit polyclonal p2x7 c terminus antibody - by Bioz Stars, 2022-08

86/100 stars

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anti p2x7 receptor antibody (Alomone Labs)

Alomone Labs anti p2x7 receptor antibody

<t>P2X7</t> receptor mediates CION-induced microglial activation and anxiodepressive-like behaviors. (A and B) Western blot analysis reveals significant upregulation of P2X7 receptor (P2X7R) level on day 14 after CION in the ipsilateral hippocampal CA1 area of rats. ** P

Anti P2x7 Receptor Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/anti p2x7 receptor antibody/product/Alomone Labs
Average 95 stars, based on 1 article reviews
Price from $9.99 to $1999.99

anti p2x7 receptor antibody - by Bioz Stars, 2022-08

95/100 stars

Buy from Supplier

Image Search Results

Journal: The Journal of Biological Chemistry

Article Title: Plasma Membrane Cholesterol as a Regulator of Human and Rodent P2X7 Receptor Activation and Sensitization *

doi: 10.1074/jbc.M114.574699

Figure Lengend Snippet: The N terminus is important for the sensitivity of P2X7 receptor activity to cholesterol depletion. A , amino acid sequence alignment of the N-terminal region of human P2X1, P2X2, and P2X7 receptors, with conserved amino acids highlighted in gray , P2X1 aa20–23 and equivalent residues in P2X2 underlined , and the P2X7 residues Lys 17 and Val 18 in red. B , time course of dye uptake mediated by the K17R/V18M mutant and wild type P2X7, alone, pretreated with MCD and loaded with cholesterol ( chol ), normalized to the untreated WT P2X7. C , bar graph of the relative rate of dye uptake normalized to the untreated WT P2X7. Dye uptake mediated by the K17R/V18M mutant is not potentiated by MCD, but is inhibited by cholesterol loading (WT: n = 14 alone, n = 11 MCD, n = 3 cholesterol; mutants: n = 12 alone, n = 11 MCD, n = 3 cholesterol; *, p

Article Snippet: Rabbit polyclonal P2X7 C terminus antibody was obtained from Alomone Labs (Jerusalem, Israel), and anti-mouse and anti-rabbit HRP-conjugated secondary antibodies were from Thermo Fisher Scientific and Bio-Rad, respectively.

Techniques: Activity Assay, Sequencing, Mutagenesis, Relative Rate

P2X7 receptor mediates CION-induced microglial activation and anxiodepressive-like behaviors. (A and B) Western blot analysis reveals significant upregulation of P2X7 receptor (P2X7R) level on day 14 after CION in the ipsilateral hippocampal CA1 area of rats. ** P

Journal: bioRxiv

Article Title: Asymmetric activation of microglia in the hippocampus drives anxiodepressive consequences of trigeminal neuralgia

doi: 10.1101/2022.04.16.488241

Figure Lengend Snippet: P2X7 receptor mediates CION-induced microglial activation and anxiodepressive-like behaviors. (A and B) Western blot analysis reveals significant upregulation of P2X7 receptor (P2X7R) level on day 14 after CION in the ipsilateral hippocampal CA1 area of rats. ** P

Article Snippet: We used the following primary antibodies: rabbit anti-P2X7R (1:2000, #APR-004, Alomone labs, Jerusalem, Israel), goat-anti IBA-1 (1:1000, #ab5076, Abcam, Cambridge, MA), and rabbit-anti IL-1β (1:500, rabbit, PeproTech, Rocky Hill, NJ).

Techniques: Activation Assay, Western Blot

P2X7 inhibition reduced the ATP release with simultaneous decrease of some pro-inflammatory cytokine levels in mouse serum and changed the redox metabolism in C6 tumors. a An ATP release is significantly lower in tumors treated with BBG compared to the control tumors ( n = 4). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Decline NOS-2 expression in BBG-treated tumors ( n = 4). c Decreased expression of FOXP3 (T reg lymphocytes) and CD68 (macrophages) markers in BBG-treated tumors evaluated using flow cytometry ( n = 5). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. d The level of the pro-inflammatory and anti-inflammatory cytokines, in mouse serum and glioma tumors using cytokine array for multiplex protein detection ( n = 3). e P2X7 inhibition caused a decrease of GSSG/GSH ratio with a slight decline of SOD activity in BBG-treated tumors ( n = 4). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

Journal: Purinergic Signalling

Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

doi: 10.1007/s11302-021-09834-2

Figure Lengend Snippet: P2X7 inhibition reduced the ATP release with simultaneous decrease of some pro-inflammatory cytokine levels in mouse serum and changed the redox metabolism in C6 tumors. a An ATP release is significantly lower in tumors treated with BBG compared to the control tumors ( n = 4). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Decline NOS-2 expression in BBG-treated tumors ( n = 4). c Decreased expression of FOXP3 (T reg lymphocytes) and CD68 (macrophages) markers in BBG-treated tumors evaluated using flow cytometry ( n = 5). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. d The level of the pro-inflammatory and anti-inflammatory cytokines, in mouse serum and glioma tumors using cytokine array for multiplex protein detection ( n = 3). e P2X7 inhibition caused a decrease of GSSG/GSH ratio with a slight decline of SOD activity in BBG-treated tumors ( n = 4). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

Article Snippet: Anti-P2X7 antibody (Alomone Labs, Israel) was diluted as recommended in the manufacturer’s instruction and incubated with tissue overnight at 4 °C.

Techniques: Inhibition, Expressing, Flow Cytometry, Multiplex Assay, Activity Assay

P2X7 receptor effects on human glioma development. a Western blot analysis of p-p38 MAPK, CD-133 and HSPA1 expression in U-138 human glioma cells ( n = 3). b In silico analysis of P2X7 expression level and survival of glioma patients using information from the TCGA database. c In silico analysis of P2X7 expression level in different WHO astrocytoma grades compared to normal tissue using NCBI dataset. d A transwell migration assay of LN-229 and U-251 glioma cells treated with 2 μM KN-62 for 24 h ( n = 3). e A crystal violet assay demonstrating a synergistic effect of 2 μM KN-62 with BCNU/TMZ co-treatment on LN-229, U-251, and U-138 glioma cell growth for 24 h ( n = 4). The significance of the differences was determined with repeated-measures one-way ANOVA with Duncan multiple range post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups

Journal: Purinergic Signalling

Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

doi: 10.1007/s11302-021-09834-2

Figure Lengend Snippet: P2X7 receptor effects on human glioma development. a Western blot analysis of p-p38 MAPK, CD-133 and HSPA1 expression in U-138 human glioma cells ( n = 3). b In silico analysis of P2X7 expression level and survival of glioma patients using information from the TCGA database. c In silico analysis of P2X7 expression level in different WHO astrocytoma grades compared to normal tissue using NCBI dataset. d A transwell migration assay of LN-229 and U-251 glioma cells treated with 2 μM KN-62 for 24 h ( n = 3). e A crystal violet assay demonstrating a synergistic effect of 2 μM KN-62 with BCNU/TMZ co-treatment on LN-229, U-251, and U-138 glioma cell growth for 24 h ( n = 4). The significance of the differences was determined with repeated-measures one-way ANOVA with Duncan multiple range post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups

Article Snippet: Anti-P2X7 antibody (Alomone Labs, Israel) was diluted as recommended in the manufacturer’s instruction and incubated with tissue overnight at 4 °C.

Techniques: Western Blot, Expressing, In Silico, Transwell Migration Assay, Crystal Violet Assay

Characterization of P2X7 receptor in glioma C6 cells in vitro. a Western blot analysis of P2X7 protein expression in C6 cells after P2X7 silencing for 72 h ( n = 3). b Calcium signal level in glioma C6 cells after P2X7 downregulation and BzATP stimulation (300 µM) ( n = 4). Gray lines represent the signals from the biological replicates; black lines represent the signal averaged among the individual replicates. Insert shows effect of P2X7 downregulation on the signal strength, one-way t -test: * P ≤ 0.05. c Measurement of bioluminescence originated from released ATP after P2X7 activation/inhibition in C6 cells ( n = 3). The statistical significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group

Journal: Purinergic Signalling

Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

doi: 10.1007/s11302-021-09834-2

Figure Lengend Snippet: Characterization of P2X7 receptor in glioma C6 cells in vitro. a Western blot analysis of P2X7 protein expression in C6 cells after P2X7 silencing for 72 h ( n = 3). b Calcium signal level in glioma C6 cells after P2X7 downregulation and BzATP stimulation (300 µM) ( n = 4). Gray lines represent the signals from the biological replicates; black lines represent the signal averaged among the individual replicates. Insert shows effect of P2X7 downregulation on the signal strength, one-way t -test: * P ≤ 0.05. c Measurement of bioluminescence originated from released ATP after P2X7 activation/inhibition in C6 cells ( n = 3). The statistical significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group

Article Snippet: Anti-P2X7 antibody (Alomone Labs, Israel) was diluted as recommended in the manufacturer’s instruction and incubated with tissue overnight at 4 °C.

Techniques: In Vitro, Western Blot, Expressing, Activation Assay, Inhibition

P2X7 activation increased C6 cell proliferation, viability, and cell adhesion in vitro. Quantitative data are presented as signal relative to control. a MTS test of long-term C6 cell viability after 100 µM BzATP stimulation of control cells and cells with P2X7 downregulation using RNAi ( n = 3). The significance of the differences was determined with paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Representative Western blot analysis of pro-survival and pro-proliferative proteins: p-p38 (Thr180/Tyr182), pAkt (Ser473), tAkt, CD133, HSPA1, and HSPA5 in C6 cell lysates after 24-h BzATP stimulation ( n = 3). c Upper panel: MTS test of C6 cell viability after 200 µM BCNU (carmustine) treatment for 24 h. Co-treatment with 100 µM BzATP and 200 µM BCNU increased viability of C6 cells ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups. Bottom panel: a crystal violet assay demonstrating a synergistic effect of BBG with BCNU/TMZ co-treatment on C6 cell growth for 24 h ( n = 6). The significance of the differences was determined with repeated measures one-way ANOVA with Duncan’s multiple range post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups. d P2X7 activation affects C6 cell adhesion to extracellular matrix components: collagen I, collagen IV, fibronectin. C6 cells showed higher adhesion potential after 24-h BzATP stimulation. 24-h co-treatment/treatment with 100 nM BBG significantly decreased C6 cell adhesion to ECM components ( n = 6). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. e P2X7 RNAi downregulation declined C6 cell adhesion to ECM components upon BzATP stimulation ( n = 4). The significance of the differences was determined with paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

Journal: Purinergic Signalling

Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

doi: 10.1007/s11302-021-09834-2

Figure Lengend Snippet: P2X7 activation increased C6 cell proliferation, viability, and cell adhesion in vitro. Quantitative data are presented as signal relative to control. a MTS test of long-term C6 cell viability after 100 µM BzATP stimulation of control cells and cells with P2X7 downregulation using RNAi ( n = 3). The significance of the differences was determined with paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Representative Western blot analysis of pro-survival and pro-proliferative proteins: p-p38 (Thr180/Tyr182), pAkt (Ser473), tAkt, CD133, HSPA1, and HSPA5 in C6 cell lysates after 24-h BzATP stimulation ( n = 3). c Upper panel: MTS test of C6 cell viability after 200 µM BCNU (carmustine) treatment for 24 h. Co-treatment with 100 µM BzATP and 200 µM BCNU increased viability of C6 cells ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups. Bottom panel: a crystal violet assay demonstrating a synergistic effect of BBG with BCNU/TMZ co-treatment on C6 cell growth for 24 h ( n = 6). The significance of the differences was determined with repeated measures one-way ANOVA with Duncan’s multiple range post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. all groups. d P2X7 activation affects C6 cell adhesion to extracellular matrix components: collagen I, collagen IV, fibronectin. C6 cells showed higher adhesion potential after 24-h BzATP stimulation. 24-h co-treatment/treatment with 100 nM BBG significantly decreased C6 cell adhesion to ECM components ( n = 6). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. e P2X7 RNAi downregulation declined C6 cell adhesion to ECM components upon BzATP stimulation ( n = 4). The significance of the differences was determined with paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

Article Snippet: Anti-P2X7 antibody (Alomone Labs, Israel) was diluted as recommended in the manufacturer’s instruction and incubated with tissue overnight at 4 °C.

Techniques: Activation Assay, In Vitro, Western Blot, Crystal Violet Assay

Effect of P2X7 inhibition using BBG on glioma C6 tumor development in vivo. a Brilliant Blue G administration led to reduction of glioma tumor mass. Representative images of control and BBG-treated C6 glioma tumors ( n = 9 for control group and n = 10 for BBG-treated group). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Representative Western blot showing the significant decrease of P2X7 expression in tumor homogenates after BBG treatment. Representative image of P2X7 immunodetection in C6 glioma tumors. c P2X7 inhibition resulted in diminished activation of matrix metalloproteinase-2 in glioma tumors. The significance of the differences was determined with Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. d Western blot analysis of known proteins involved in cell adhesion and EMT signaling in C6 tumor homogenates ( n = 4). e Western blot analysis of known proteins involved in tumor aggressiveness in C6 tumor homogenates ( n = 4)

Journal: Purinergic Signalling

Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

doi: 10.1007/s11302-021-09834-2

Figure Lengend Snippet: Effect of P2X7 inhibition using BBG on glioma C6 tumor development in vivo. a Brilliant Blue G administration led to reduction of glioma tumor mass. Representative images of control and BBG-treated C6 glioma tumors ( n = 9 for control group and n = 10 for BBG-treated group). The significance of the differences was determined using Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. b Representative Western blot showing the significant decrease of P2X7 expression in tumor homogenates after BBG treatment. Representative image of P2X7 immunodetection in C6 glioma tumors. c P2X7 inhibition resulted in diminished activation of matrix metalloproteinase-2 in glioma tumors. The significance of the differences was determined with Student’s t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. d Western blot analysis of known proteins involved in cell adhesion and EMT signaling in C6 tumor homogenates ( n = 4). e Western blot analysis of known proteins involved in tumor aggressiveness in C6 tumor homogenates ( n = 4)

Article Snippet: Anti-P2X7 antibody (Alomone Labs, Israel) was diluted as recommended in the manufacturer’s instruction and incubated with tissue overnight at 4 °C.

Techniques: Inhibition, In Vivo, Western Blot, Expressing, Immunodetection, Activation Assay

Activation of P2X7 receptor led to elevated ROS production and mitochondrial membrane depolarization in C6 cells. Quantitative data are presented as signal relative to control. a DCF-DA fluorescence measurement in C6 glioma cells. Cells stimulated with 100 µM BzATP for 1 h were characterized by increased ROS production compared to control in C6 cells ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. b Potential-dependent staining of mitochondria in C6 cells using JC-1. 1-h BzATP (100 µM) stimulation led to considerable mitochondrial membrane depolarization ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. c Representative images of MitoTracker Red CMXRos accumulation in C6 cells stimulated with 100 µM BzATP for 1 h. d DCF-DA fluorescence measurement in C6 glioma cells with RNA interference of P2X7 expression ( n = 4). Downregulation of P2X7 led to decreased ROS production in C6 cells after 1-h BzATP (100 µM) stimulation. The significance of the differences was determined using paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. e Potential-dependent staining of mitochondria using JC-1 dye in C6 cells with RNA interference of P2X7 expression after 1 h of 100 μM BzATP pre-treatment ( n = 3). The amount of depolarized mitochondria was higher in control cells when compared to that in cells with P2X7 downregulation. The significance of the differences was determined using paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

Journal: Purinergic Signalling

Article Title: P2X7 receptor: the regulator of glioma tumor development and survival

doi: 10.1007/s11302-021-09834-2

Figure Lengend Snippet: Activation of P2X7 receptor led to elevated ROS production and mitochondrial membrane depolarization in C6 cells. Quantitative data are presented as signal relative to control. a DCF-DA fluorescence measurement in C6 glioma cells. Cells stimulated with 100 µM BzATP for 1 h were characterized by increased ROS production compared to control in C6 cells ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. b Potential-dependent staining of mitochondria in C6 cells using JC-1. 1-h BzATP (100 µM) stimulation led to considerable mitochondrial membrane depolarization ( n = 3). The significance of the differences was determined with one-way ANOVA with Bonferroni post hoc test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. control group. c Representative images of MitoTracker Red CMXRos accumulation in C6 cells stimulated with 100 µM BzATP for 1 h. d DCF-DA fluorescence measurement in C6 glioma cells with RNA interference of P2X7 expression ( n = 4). Downregulation of P2X7 led to decreased ROS production in C6 cells after 1-h BzATP (100 µM) stimulation. The significance of the differences was determined using paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control. e Potential-dependent staining of mitochondria using JC-1 dye in C6 cells with RNA interference of P2X7 expression after 1 h of 100 μM BzATP pre-treatment ( n = 3). The amount of depolarized mitochondria was higher in control cells when compared to that in cells with P2X7 downregulation. The significance of the differences was determined using paired t -test: * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001 vs. the respective control

Article Snippet: Anti-P2X7 antibody (Alomone Labs, Israel) was diluted as recommended in the manufacturer’s instruction and incubated with tissue overnight at 4 °C.

Techniques: Activation Assay, Fluorescence, Staining, Expressing

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

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

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:

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: