p2x7 intracellular epitope antibody  (Alomone Labs)


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    Alomone Labs p2x7 intracellular epitope antibody
    <t>P2X7</t> receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.
    P2x7 Intracellular Epitope Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    p2x7 intracellular epitope antibody - by Bioz Stars, 2022-06
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    Images

    1) Product Images from "Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis"

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01529

    P2X7 receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.
    Figure Legend Snippet: P2X7 receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.

    Techniques Used: Expressing, Immunofluorescence

    Monocyte chemoattractant protein-1 (MCP-1) chemokine colocalizes with P2X7 receptor (P2X7R) on astrocytes in secondary progressive multiple sclerosis (SPMS) frontal cortex. Triple immunofluorescence confocal analysis performed on sections of frontal cortex from control (A–D) and SPMS (E–H) with antibodies for P2X7R [ (A,E) , red], MCP-1 [ (B,F) , green], and glial fibrillary acidic protein [ (C,G) , blue] shows colocalization [ (D,H) , white signal] and strong up-regulation of signals in white matter of SPMS (E–H) .
    Figure Legend Snippet: Monocyte chemoattractant protein-1 (MCP-1) chemokine colocalizes with P2X7 receptor (P2X7R) on astrocytes in secondary progressive multiple sclerosis (SPMS) frontal cortex. Triple immunofluorescence confocal analysis performed on sections of frontal cortex from control (A–D) and SPMS (E–H) with antibodies for P2X7R [ (A,E) , red], MCP-1 [ (B,F) , green], and glial fibrillary acidic protein [ (C,G) , blue] shows colocalization [ (D,H) , white signal] and strong up-regulation of signals in white matter of SPMS (E–H) .

    Techniques Used: Immunofluorescence

    Spatiotemporal profile of P2X7 receptor (P2X7R) expression in multiple sclerosis (MS). The cartoon describes that P2X7R is down-regulated in monocytes during their activation and extravasation from blood vessel to MS cerebral cortex, while the receptor and the monocyte chemoattractant protein-1 chemokine are up-regulated on MS astrocytes concurrently with increased glial fibrillary acidic protein signal and glial scar formation.
    Figure Legend Snippet: Spatiotemporal profile of P2X7 receptor (P2X7R) expression in multiple sclerosis (MS). The cartoon describes that P2X7R is down-regulated in monocytes during their activation and extravasation from blood vessel to MS cerebral cortex, while the receptor and the monocyte chemoattractant protein-1 chemokine are up-regulated on MS astrocytes concurrently with increased glial fibrillary acidic protein signal and glial scar formation.

    Techniques Used: Expressing, Activation Assay

    P2X7 receptor (P2X7R) is present on monocytes in blood vessels of secondary progressive multiple sclerosis (SPMS) frontal cortex. (A) Immunohistochemistry on sections from human frontal cortex shows roundish P2X7R-positive cells distributed within distinct clusters throughout the entire SPMS tissue. Confocal double immunofluorescence analysis of these clusters exhibits colocalization of P2X7R protein (red) with CD45 leukocyte marker [ (B) , green]. Staining with Lectin from Lycopersicon esculentum for vascular endothelial cells [ (C,D) , green] clearly shows the presence of P2X7R-positive roundish cells (red) within the lumen of blood vessels (asterisk). Double immunofluorescence of P2X7R-positive clusters shows colocalization of P2X7R (red) with cluster of differentiation 14 (CD14) monocyte/macrophage marker [ (E) , green]. Confocal triple immunofluorescence analysis is then performed with antibodies for CD14 [ (F,G) , green], P2X7R [ (F,G) , red], and microglia/macrophages markers CD68 (F) or major histocompatibility complex II [ (G) , blue]. The asterisk always indicates the lumen of blood vessels, arrows indicate P2X7R-negative cells, and arrowheads P2X7R-positive cells.
    Figure Legend Snippet: P2X7 receptor (P2X7R) is present on monocytes in blood vessels of secondary progressive multiple sclerosis (SPMS) frontal cortex. (A) Immunohistochemistry on sections from human frontal cortex shows roundish P2X7R-positive cells distributed within distinct clusters throughout the entire SPMS tissue. Confocal double immunofluorescence analysis of these clusters exhibits colocalization of P2X7R protein (red) with CD45 leukocyte marker [ (B) , green]. Staining with Lectin from Lycopersicon esculentum for vascular endothelial cells [ (C,D) , green] clearly shows the presence of P2X7R-positive roundish cells (red) within the lumen of blood vessels (asterisk). Double immunofluorescence of P2X7R-positive clusters shows colocalization of P2X7R (red) with cluster of differentiation 14 (CD14) monocyte/macrophage marker [ (E) , green]. Confocal triple immunofluorescence analysis is then performed with antibodies for CD14 [ (F,G) , green], P2X7R [ (F,G) , red], and microglia/macrophages markers CD68 (F) or major histocompatibility complex II [ (G) , blue]. The asterisk always indicates the lumen of blood vessels, arrows indicate P2X7R-negative cells, and arrowheads P2X7R-positive cells.

    Techniques Used: Immunohistochemistry, Immunofluorescence, Marker, Staining

    P2X7 receptor (P2X7R) is present on astrocytes in the parenchyma of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal analysis of SPMS frontal cortex parenchyma shows absence of colocalization of P2X7R (red) with P2Y12R [ (A) , green], major histocompatibility complex II (MHC II) [ (B) , blue], and myelin basic protein [ (C) , blue], but the presence of merged P2X7R/glial fibrillary acidic protein signals (D–F) . P2X7R/MHC II-positive signal is also visible but confined in the lumen of a blood vessel [ (B) , arrow, pink]. Immunohistochemistry analysis of cortical parenchyma reveals the abundant presence of P2X7R in GM on interlaminar (G) and protoplasmic astrocytes (E) , and in white matter on fibrous astrocytes (I,J) . In (J) , astrocytic “vascular feet” are visible adjacent to the blood vessel walls (arrows).
    Figure Legend Snippet: P2X7 receptor (P2X7R) is present on astrocytes in the parenchyma of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal analysis of SPMS frontal cortex parenchyma shows absence of colocalization of P2X7R (red) with P2Y12R [ (A) , green], major histocompatibility complex II (MHC II) [ (B) , blue], and myelin basic protein [ (C) , blue], but the presence of merged P2X7R/glial fibrillary acidic protein signals (D–F) . P2X7R/MHC II-positive signal is also visible but confined in the lumen of a blood vessel [ (B) , arrow, pink]. Immunohistochemistry analysis of cortical parenchyma reveals the abundant presence of P2X7R in GM on interlaminar (G) and protoplasmic astrocytes (E) , and in white matter on fibrous astrocytes (I,J) . In (J) , astrocytic “vascular feet” are visible adjacent to the blood vessel walls (arrows).

    Techniques Used: Immunohistochemistry

    P2X7 receptor (P2X7R) immunoreactivity is increased with astrogliosis in white matter (WM) of secondary progressive multiple sclerosis (SPMS) frontal cortex parenchyma. Immunohistochemistry analysis of two adjacent WM fields from SPMS frontal cortex characterized, respectively, by hypertrophic astrocytes (B) and glial scar (C) reveals a noteworthy increase in P2X7R-positive astrocytes (B,C) , compared with control case (A) .
    Figure Legend Snippet: P2X7 receptor (P2X7R) immunoreactivity is increased with astrogliosis in white matter (WM) of secondary progressive multiple sclerosis (SPMS) frontal cortex parenchyma. Immunohistochemistry analysis of two adjacent WM fields from SPMS frontal cortex characterized, respectively, by hypertrophic astrocytes (B) and glial scar (C) reveals a noteworthy increase in P2X7R-positive astrocytes (B,C) , compared with control case (A) .

    Techniques Used: Immunohistochemistry

    P2X7 receptor (P2X7R) mRNA and protein are augmented in secondary progressive multiple sclerosis. (A) Total RNA was extracted from three different snap-frozen blocks from MS patients (cases MS114, MS125, and MS163) and six controls (cases C12–101, C12–112, C13–010, C13–022, C14–069, and C14–053) and the expression of P2X7R mRNA was examined by RT-qPCR. Cortical protein extracts (15 μg/well) from different tissue blocks (A5C3, A5B3, A3B1, A1A2, A2A1, and A2B2) of MS cases MS114, MS125, and MS163 were analyzed by western blotting for the modulation of P2X7R, with respect to control cases (C12–101, C12–112, C13–010, C13–022, and C14–069), in both detergent-soluble (B) and -insoluble fractions (C) . GAPDH was used for protein normalization. Statistical significance was calculated by Student’s t-test, * p
    Figure Legend Snippet: P2X7 receptor (P2X7R) mRNA and protein are augmented in secondary progressive multiple sclerosis. (A) Total RNA was extracted from three different snap-frozen blocks from MS patients (cases MS114, MS125, and MS163) and six controls (cases C12–101, C12–112, C13–010, C13–022, C14–069, and C14–053) and the expression of P2X7R mRNA was examined by RT-qPCR. Cortical protein extracts (15 μg/well) from different tissue blocks (A5C3, A5B3, A3B1, A1A2, A2A1, and A2B2) of MS cases MS114, MS125, and MS163 were analyzed by western blotting for the modulation of P2X7R, with respect to control cases (C12–101, C12–112, C13–010, C13–022, and C14–069), in both detergent-soluble (B) and -insoluble fractions (C) . GAPDH was used for protein normalization. Statistical significance was calculated by Student’s t-test, * p

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

    P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) monocytes and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive cells within freshly isolated peripheral blood mononuclear cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p
    Figure Legend Snippet: P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) monocytes and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive cells within freshly isolated peripheral blood mononuclear cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p

    Techniques Used: In Vitro, Quantitative RT-PCR, Isolation, Flow Cytometry

    2) Product Images from "Differential expression of P2X7 receptor and IL-1β in nociceptive and neuropathic pain"

    Article Title: Differential expression of P2X7 receptor and IL-1β in nociceptive and neuropathic pain

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-016-0565-z

    P2X7R expression on lymphocytes ( a ), CD4 + ( b ) and CD4 − cells ( c ), and monocytes ( d ) is elevated in patients with neuropathic pain. PBMCs were stained with PerCP-labeled antihuman CD4 antibody and FITC-labeled antihuman P2X7R antibody and analyzed by FACS. The results show a significantly elevated P2X7R expression only in patients suffering from neuropathic pain on the analyzed cell type (lymphocytes ( p = 0.009), CD4 + cells ( p
    Figure Legend Snippet: P2X7R expression on lymphocytes ( a ), CD4 + ( b ) and CD4 − cells ( c ), and monocytes ( d ) is elevated in patients with neuropathic pain. PBMCs were stained with PerCP-labeled antihuman CD4 antibody and FITC-labeled antihuman P2X7R antibody and analyzed by FACS. The results show a significantly elevated P2X7R expression only in patients suffering from neuropathic pain on the analyzed cell type (lymphocytes ( p = 0.009), CD4 + cells ( p

    Techniques Used: Expressing, Staining, Labeling, FACS

    IL-1β levels are elevated in patients with neuropathic pain. As IL-1β was shown to be the key mediator in P2X7R-pain interplay, we analyzed serum levels of this pro-inflammatory and pro-nociceptive cytokine by multiplex immune assay. Affirmatively to the results with elevated P2X7Rs, we solely found significant increased serum levels in the peripheral blood of patients with NeP * p
    Figure Legend Snippet: IL-1β levels are elevated in patients with neuropathic pain. As IL-1β was shown to be the key mediator in P2X7R-pain interplay, we analyzed serum levels of this pro-inflammatory and pro-nociceptive cytokine by multiplex immune assay. Affirmatively to the results with elevated P2X7Rs, we solely found significant increased serum levels in the peripheral blood of patients with NeP * p

    Techniques Used: Multiplex Assay

    Exemplary illustration of P2X7 receptor expression. For quantitative analysis of the proportion of CD4 + cells ( a ) and P2X7R protein expression ( b ), cells were stained with PerCP-labeled antihuman CD4 antibody and FITC-labeled antihuman P2X7R antibody. We separately analyzed each subgroup by the mean fluorescence intensity (MFI) of P2X7R expression. Overlay histograms of representative results of P2X7R expression of one patient/healthy volunteer are displayed in histograms
    Figure Legend Snippet: Exemplary illustration of P2X7 receptor expression. For quantitative analysis of the proportion of CD4 + cells ( a ) and P2X7R protein expression ( b ), cells were stained with PerCP-labeled antihuman CD4 antibody and FITC-labeled antihuman P2X7R antibody. We separately analyzed each subgroup by the mean fluorescence intensity (MFI) of P2X7R expression. Overlay histograms of representative results of P2X7R expression of one patient/healthy volunteer are displayed in histograms

    Techniques Used: Expressing, Staining, Labeling, Fluorescence

    P2RX7 mRNA expression is exclusively increased in patients with neuropathic pain. To confirm the flow cytometric observations with elevated P2X7R protein expression, predominantly on CD4 + cells and only in NeP, we determined the relative P2RX7 mRNA expression of CD4 + cells by qPCR. Affirmatively, increased mRNA levels were consistent with the flow cytometric analyses and exclusively elevated in patients with NeP. ( p = 0.038). * p
    Figure Legend Snippet: P2RX7 mRNA expression is exclusively increased in patients with neuropathic pain. To confirm the flow cytometric observations with elevated P2X7R protein expression, predominantly on CD4 + cells and only in NeP, we determined the relative P2RX7 mRNA expression of CD4 + cells by qPCR. Affirmatively, increased mRNA levels were consistent with the flow cytometric analyses and exclusively elevated in patients with NeP. ( p = 0.038). * p

    Techniques Used: Expressing, Real-time Polymerase Chain Reaction

    Differential blood count and quantification of CD4 + cells. In order to avoid misinterpretation of potentially elevated P2X7R protein expression based on different cell counts, we quantified numbers of polymorphonuclear leukocytes ( a ), lymphocytes ( b ), and CD4 + cells ( c ). No differences were found between patients suffering from CLBP, NeP, and healthy volunteers. Data are presented as mean ± SD, n = 19, * p
    Figure Legend Snippet: Differential blood count and quantification of CD4 + cells. In order to avoid misinterpretation of potentially elevated P2X7R protein expression based on different cell counts, we quantified numbers of polymorphonuclear leukocytes ( a ), lymphocytes ( b ), and CD4 + cells ( c ). No differences were found between patients suffering from CLBP, NeP, and healthy volunteers. Data are presented as mean ± SD, n = 19, * p

    Techniques Used: Expressing

    3) Product Images from "Early P2X7R-dependent activation of microglia during the asymptomatic phase of autoimmune encephalomyelitis"

    Article Title: Early P2X7R-dependent activation of microglia during the asymptomatic phase of autoimmune encephalomyelitis

    Journal: Inflammopharmacology

    doi: 10.1007/s10787-018-0528-3

    Activation of microglia during the course of EAE is suppressed by the P2X7R antagonist. Immunofluorescence image of Iba-1immunoreactive cells in forebrain of control ( a ), EAE rats ( b , c ) and EAE rats treated with BBG ( d , e ) in asymptomatic (4 d.p.i.) and symptomatic (12 d.p.i.) phases. Insets present magnification of resting ( a ′, e ′) and activated ( c ′) microglial cells. The graph indicates the mean intensity of the fluorescence signal. *** p
    Figure Legend Snippet: Activation of microglia during the course of EAE is suppressed by the P2X7R antagonist. Immunofluorescence image of Iba-1immunoreactive cells in forebrain of control ( a ), EAE rats ( b , c ) and EAE rats treated with BBG ( d , e ) in asymptomatic (4 d.p.i.) and symptomatic (12 d.p.i.) phases. Insets present magnification of resting ( a ′, e ′) and activated ( c ′) microglial cells. The graph indicates the mean intensity of the fluorescence signal. *** p

    Techniques Used: Activation Assay, Immunofluorescence, Fluorescence

    Expression of P2X7R in microglial cells. Immunofluorescence image of double immunostaining Iba-1/P2X7R in forebrain of control ( a ) and EAE rats in asymptomatic (4 d.p.i.) ( b ) and symptomatic (12 d.p.i.) ( c ) phases of the disease. Activated Iba-1/P2X7R-positive cells are seen in immunized rats ( b , c ). Images are representative for each of four animals
    Figure Legend Snippet: Expression of P2X7R in microglial cells. Immunofluorescence image of double immunostaining Iba-1/P2X7R in forebrain of control ( a ) and EAE rats in asymptomatic (4 d.p.i.) ( b ) and symptomatic (12 d.p.i.) ( c ) phases of the disease. Activated Iba-1/P2X7R-positive cells are seen in immunized rats ( b , c ). Images are representative for each of four animals

    Techniques Used: Expressing, Immunofluorescence, Double Immunostaining

    4) Product Images from "Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice"

    Article Title: Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

    Journal: BMC Cancer

    doi: 10.1186/1471-2407-9-114

    Effects of anti-sense P2X 7 oligonucleotides on BzATP-induced increase in cytosolic calcium (ΔCa 2+ i , A) and on the influx of ethidium bromide (Eth-Br, B) . Cultured mouse wild-type normal keratinocytes were pre-treated with 100 μM anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) for 14 hours followed by 8 hours treatment with 100 μM BzATP. Control – cells treated with the vehicle of the ASO. Levels of ΔCa 2+ i ( A ) and of Eth-Br fluorescence ( B ) were determined as in Fig. 12. The experiments were repeated twice with similar trends.
    Figure Legend Snippet: Effects of anti-sense P2X 7 oligonucleotides on BzATP-induced increase in cytosolic calcium (ΔCa 2+ i , A) and on the influx of ethidium bromide (Eth-Br, B) . Cultured mouse wild-type normal keratinocytes were pre-treated with 100 μM anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) for 14 hours followed by 8 hours treatment with 100 μM BzATP. Control – cells treated with the vehicle of the ASO. Levels of ΔCa 2+ i ( A ) and of Eth-Br fluorescence ( B ) were determined as in Fig. 12. The experiments were repeated twice with similar trends.

    Techniques Used: Cell Culture, Allele-specific Oligonucleotide, Fluorescence

    Effects of pre-treatment with anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) (both at 100 μM for 14 hours), and of treatments with BzATP (100 μM, 8 hours) on [ 3 H]thymidine incorporation in mouse wild-type normal keratinocytes (values are means ± SD, n = 4) .
    Figure Legend Snippet: Effects of pre-treatment with anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) (both at 100 μM for 14 hours), and of treatments with BzATP (100 μM, 8 hours) on [ 3 H]thymidine incorporation in mouse wild-type normal keratinocytes (values are means ± SD, n = 4) .

    Techniques Used: Allele-specific Oligonucleotide

    Effects of BzATP on apoptosis in cultured mouse keratinocytes . In all experiments levels of apoptosis were normalized to an arbitrary value of 2 in control cells. AU – arbitrary units. A . BzATP dose-response effect in cultured mouse normal (wild-type, C57Bl) keratinocytes (means ± SD, n = 3). Cells were treated with one of the indicated concentrations of BzATP for 8 hours. B . Cultured mouse normal keratinocytes (wild-type, C57Bl) were pre-treated with 100 μM anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) for 14 hours followed by 8 hours treatment with 100 μM BzATP. Control – cells treated with the vehicle of the ASO. Values are means (± SD) of 3 experiments for each condition. Insert in B is Western immunoblot with anti-P2X 7 antibody of lysates of cells treated with ASO or RCO (n = 2). C . BzATP time-response effect in cultured mouse normal keratinocytes (wild-type, C57Bl; filled triangles), or in keratinocytes obtained from P2X 7 -deficient (P2X 7 -/-Pfizer [empty circles] or P2X 7 -/-GSK [filled circles]) mice (both in the C57Bl background). Values are means (± SD) of 3 experiments for each condition. Changes in apoptosis in A and B were determined in terms of solubilized DNA; changes in apoptosis in C were determined using cell-death detection ELISA.
    Figure Legend Snippet: Effects of BzATP on apoptosis in cultured mouse keratinocytes . In all experiments levels of apoptosis were normalized to an arbitrary value of 2 in control cells. AU – arbitrary units. A . BzATP dose-response effect in cultured mouse normal (wild-type, C57Bl) keratinocytes (means ± SD, n = 3). Cells were treated with one of the indicated concentrations of BzATP for 8 hours. B . Cultured mouse normal keratinocytes (wild-type, C57Bl) were pre-treated with 100 μM anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) for 14 hours followed by 8 hours treatment with 100 μM BzATP. Control – cells treated with the vehicle of the ASO. Values are means (± SD) of 3 experiments for each condition. Insert in B is Western immunoblot with anti-P2X 7 antibody of lysates of cells treated with ASO or RCO (n = 2). C . BzATP time-response effect in cultured mouse normal keratinocytes (wild-type, C57Bl; filled triangles), or in keratinocytes obtained from P2X 7 -deficient (P2X 7 -/-Pfizer [empty circles] or P2X 7 -/-GSK [filled circles]) mice (both in the C57Bl background). Values are means (± SD) of 3 experiments for each condition. Changes in apoptosis in A and B were determined in terms of solubilized DNA; changes in apoptosis in C were determined using cell-death detection ELISA.

    Techniques Used: Cell Culture, Allele-specific Oligonucleotide, Western Blot, Mouse Assay, Enzyme-linked Immunosorbent Assay

    P2X 7 immunoreactivity in mouse normal skin (A), papilloma (C), and skin cancer tissues (E) (Experiment-1); B, D, F are parallel cross sections, respectively, stained by H E . G . Analysis of P2X 7 immunoreactivity compared among paired histologically normal and cancerous tissues. Bars are means (± SD) of levels in tissues of five mice. H . P2X 7 protein assays in mouse normal and cancer skin tissues. Lysates fractionated by gel electrophoresis were immunoblotted with the anti P2X 7 antibody and membranes were reprobed with the anti GAPDH antibody. Insert in H shows Western immunoblot of lysates of histologically normal and cancerous tissues obtained from the same animal. Similar results were obtained in tissues of two additional mice. Bars show means (± SD) of densitometry results of the P2X 7 -specific 75 KDa bands in tissues of three mice. I . P2X 7 mRNA levels (relative to GAPDH mRNA) (means ± SD) in histologically normal and cancerous tissues obtained from three animals. In G-I data in the normal tissues were normalized in each case to an arbitrary value of 10. * – p
    Figure Legend Snippet: P2X 7 immunoreactivity in mouse normal skin (A), papilloma (C), and skin cancer tissues (E) (Experiment-1); B, D, F are parallel cross sections, respectively, stained by H E . G . Analysis of P2X 7 immunoreactivity compared among paired histologically normal and cancerous tissues. Bars are means (± SD) of levels in tissues of five mice. H . P2X 7 protein assays in mouse normal and cancer skin tissues. Lysates fractionated by gel electrophoresis were immunoblotted with the anti P2X 7 antibody and membranes were reprobed with the anti GAPDH antibody. Insert in H shows Western immunoblot of lysates of histologically normal and cancerous tissues obtained from the same animal. Similar results were obtained in tissues of two additional mice. Bars show means (± SD) of densitometry results of the P2X 7 -specific 75 KDa bands in tissues of three mice. I . P2X 7 mRNA levels (relative to GAPDH mRNA) (means ± SD) in histologically normal and cancerous tissues obtained from three animals. In G-I data in the normal tissues were normalized in each case to an arbitrary value of 10. * – p

    Techniques Used: Staining, Mouse Assay, Nucleic Acid Electrophoresis, Western Blot

    Effects in mice in-vivo of local treatment with BzATP on skin apoptosis (Experiment-3) . Animals (n = 5) were treated for 16 weeks either with BzATP, applied locally twice a week on the shaved dorsal skin ( A, B ) or with the vehicle (Control, n = 5, C, D ). At the end of the experiment animals were euthanized and strips were obtained from each animal dorsal skin areas for H E ( B, D ) and P2X 7 /TUNEL co-staining ( E-L ). Arrows in J show increased TUNEL staining co-localizing with P2X 7 immunoreactivity in epidermal cells of the basal/parabasal layers (horizontal arrow) and of epidermal hair shaft cells (vertical arrow). Assays were repeated 3–5 times with similar trends.
    Figure Legend Snippet: Effects in mice in-vivo of local treatment with BzATP on skin apoptosis (Experiment-3) . Animals (n = 5) were treated for 16 weeks either with BzATP, applied locally twice a week on the shaved dorsal skin ( A, B ) or with the vehicle (Control, n = 5, C, D ). At the end of the experiment animals were euthanized and strips were obtained from each animal dorsal skin areas for H E ( B, D ) and P2X 7 /TUNEL co-staining ( E-L ). Arrows in J show increased TUNEL staining co-localizing with P2X 7 immunoreactivity in epidermal cells of the basal/parabasal layers (horizontal arrow) and of epidermal hair shaft cells (vertical arrow). Assays were repeated 3–5 times with similar trends.

    Techniques Used: Mouse Assay, In Vivo, TUNEL Assay, Staining

    Effects of treatments with BzATP on P2X 7 expression and apoptosis in DMBA/TPA – induced skin papillomas (A-D) and cancers (E-H) (Experiment-1) . Assays were repeated 4 times with similar trends. C, D, G, H are parallel cross sections to A, B, E, F , respectively.
    Figure Legend Snippet: Effects of treatments with BzATP on P2X 7 expression and apoptosis in DMBA/TPA – induced skin papillomas (A-D) and cancers (E-H) (Experiment-1) . Assays were repeated 4 times with similar trends. C, D, G, H are parallel cross sections to A, B, E, F , respectively.

    Techniques Used: Expressing

    5) Product Images from "Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis"

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    Journal: Frontiers in Immunology

    doi: 10.3389/fimmu.2017.01529

    P2X7 receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.
    Figure Legend Snippet: P2X7 receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.

    Techniques Used: Expressing, Immunofluorescence

    Monocyte chemoattractant protein-1 (MCP-1) chemokine colocalizes with P2X7 receptor (P2X7R) on astrocytes in secondary progressive multiple sclerosis (SPMS) frontal cortex. Triple immunofluorescence confocal analysis performed on sections of frontal cortex from control (A–D) and SPMS (E–H) with antibodies for P2X7R [ (A,E) , red], MCP-1 [ (B,F) , green], and glial fibrillary acidic protein [ (C,G) , blue] shows colocalization [ (D,H) , white signal] and strong up-regulation of signals in white matter of SPMS (E–H) .
    Figure Legend Snippet: Monocyte chemoattractant protein-1 (MCP-1) chemokine colocalizes with P2X7 receptor (P2X7R) on astrocytes in secondary progressive multiple sclerosis (SPMS) frontal cortex. Triple immunofluorescence confocal analysis performed on sections of frontal cortex from control (A–D) and SPMS (E–H) with antibodies for P2X7R [ (A,E) , red], MCP-1 [ (B,F) , green], and glial fibrillary acidic protein [ (C,G) , blue] shows colocalization [ (D,H) , white signal] and strong up-regulation of signals in white matter of SPMS (E–H) .

    Techniques Used: Immunofluorescence

    Spatiotemporal profile of P2X7 receptor (P2X7R) expression in multiple sclerosis (MS). The cartoon describes that P2X7R is down-regulated in monocytes during their activation and extravasation from blood vessel to MS cerebral cortex, while the receptor and the monocyte chemoattractant protein-1 chemokine are up-regulated on MS astrocytes concurrently with increased glial fibrillary acidic protein signal and glial scar formation.
    Figure Legend Snippet: Spatiotemporal profile of P2X7 receptor (P2X7R) expression in multiple sclerosis (MS). The cartoon describes that P2X7R is down-regulated in monocytes during their activation and extravasation from blood vessel to MS cerebral cortex, while the receptor and the monocyte chemoattractant protein-1 chemokine are up-regulated on MS astrocytes concurrently with increased glial fibrillary acidic protein signal and glial scar formation.

    Techniques Used: Expressing, Activation Assay

    P2X7 receptor (P2X7R) is present on monocytes in blood vessels of secondary progressive multiple sclerosis (SPMS) frontal cortex. (A) Immunohistochemistry on sections from human frontal cortex shows roundish P2X7R-positive cells distributed within distinct clusters throughout the entire SPMS tissue. Confocal double immunofluorescence analysis of these clusters exhibits colocalization of P2X7R protein (red) with CD45 leukocyte marker [ (B) , green]. Staining with Lectin from Lycopersicon esculentum for vascular endothelial cells [ (C,D) , green] clearly shows the presence of P2X7R-positive roundish cells (red) within the lumen of blood vessels (asterisk). Double immunofluorescence of P2X7R-positive clusters shows colocalization of P2X7R (red) with cluster of differentiation 14 (CD14) monocyte/macrophage marker [ (E) , green]. Confocal triple immunofluorescence analysis is then performed with antibodies for CD14 [ (F,G) , green], P2X7R [ (F,G) , red], and microglia/macrophages markers CD68 (F) or major histocompatibility complex II [ (G) , blue]. The asterisk always indicates the lumen of blood vessels, arrows indicate P2X7R-negative cells, and arrowheads P2X7R-positive cells.
    Figure Legend Snippet: P2X7 receptor (P2X7R) is present on monocytes in blood vessels of secondary progressive multiple sclerosis (SPMS) frontal cortex. (A) Immunohistochemistry on sections from human frontal cortex shows roundish P2X7R-positive cells distributed within distinct clusters throughout the entire SPMS tissue. Confocal double immunofluorescence analysis of these clusters exhibits colocalization of P2X7R protein (red) with CD45 leukocyte marker [ (B) , green]. Staining with Lectin from Lycopersicon esculentum for vascular endothelial cells [ (C,D) , green] clearly shows the presence of P2X7R-positive roundish cells (red) within the lumen of blood vessels (asterisk). Double immunofluorescence of P2X7R-positive clusters shows colocalization of P2X7R (red) with cluster of differentiation 14 (CD14) monocyte/macrophage marker [ (E) , green]. Confocal triple immunofluorescence analysis is then performed with antibodies for CD14 [ (F,G) , green], P2X7R [ (F,G) , red], and microglia/macrophages markers CD68 (F) or major histocompatibility complex II [ (G) , blue]. The asterisk always indicates the lumen of blood vessels, arrows indicate P2X7R-negative cells, and arrowheads P2X7R-positive cells.

    Techniques Used: Immunohistochemistry, Immunofluorescence, Marker, Staining

    P2X7 receptor (P2X7R) is present on astrocytes in the parenchyma of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal analysis of SPMS frontal cortex parenchyma shows absence of colocalization of P2X7R (red) with P2Y12R [ (A) , green], major histocompatibility complex II (MHC II) [ (B) , blue], and myelin basic protein [ (C) , blue], but the presence of merged P2X7R/glial fibrillary acidic protein signals (D–F) . P2X7R/MHC II-positive signal is also visible but confined in the lumen of a blood vessel [ (B) , arrow, pink]. Immunohistochemistry analysis of cortical parenchyma reveals the abundant presence of P2X7R in GM on interlaminar (G) and protoplasmic astrocytes (E) , and in white matter on fibrous astrocytes (I,J) . In (J) , astrocytic “vascular feet” are visible adjacent to the blood vessel walls (arrows).
    Figure Legend Snippet: P2X7 receptor (P2X7R) is present on astrocytes in the parenchyma of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal analysis of SPMS frontal cortex parenchyma shows absence of colocalization of P2X7R (red) with P2Y12R [ (A) , green], major histocompatibility complex II (MHC II) [ (B) , blue], and myelin basic protein [ (C) , blue], but the presence of merged P2X7R/glial fibrillary acidic protein signals (D–F) . P2X7R/MHC II-positive signal is also visible but confined in the lumen of a blood vessel [ (B) , arrow, pink]. Immunohistochemistry analysis of cortical parenchyma reveals the abundant presence of P2X7R in GM on interlaminar (G) and protoplasmic astrocytes (E) , and in white matter on fibrous astrocytes (I,J) . In (J) , astrocytic “vascular feet” are visible adjacent to the blood vessel walls (arrows).

    Techniques Used: Immunohistochemistry

    P2X7 receptor (P2X7R) immunoreactivity is increased with astrogliosis in white matter (WM) of secondary progressive multiple sclerosis (SPMS) frontal cortex parenchyma. Immunohistochemistry analysis of two adjacent WM fields from SPMS frontal cortex characterized, respectively, by hypertrophic astrocytes (B) and glial scar (C) reveals a noteworthy increase in P2X7R-positive astrocytes (B,C) , compared with control case (A) .
    Figure Legend Snippet: P2X7 receptor (P2X7R) immunoreactivity is increased with astrogliosis in white matter (WM) of secondary progressive multiple sclerosis (SPMS) frontal cortex parenchyma. Immunohistochemistry analysis of two adjacent WM fields from SPMS frontal cortex characterized, respectively, by hypertrophic astrocytes (B) and glial scar (C) reveals a noteworthy increase in P2X7R-positive astrocytes (B,C) , compared with control case (A) .

    Techniques Used: Immunohistochemistry

    P2X7 receptor (P2X7R) mRNA and protein are augmented in secondary progressive multiple sclerosis. (A) Total RNA was extracted from three different snap-frozen blocks from MS patients (cases MS114, MS125, and MS163) and six controls (cases C12–101, C12–112, C13–010, C13–022, C14–069, and C14–053) and the expression of P2X7R mRNA was examined by RT-qPCR. Cortical protein extracts (15 μg/well) from different tissue blocks (A5C3, A5B3, A3B1, A1A2, A2A1, and A2B2) of MS cases MS114, MS125, and MS163 were analyzed by western blotting for the modulation of P2X7R, with respect to control cases (C12–101, C12–112, C13–010, C13–022, and C14–069), in both detergent-soluble (B) and -insoluble fractions (C) . GAPDH was used for protein normalization. Statistical significance was calculated by Student’s t-test, * p
    Figure Legend Snippet: P2X7 receptor (P2X7R) mRNA and protein are augmented in secondary progressive multiple sclerosis. (A) Total RNA was extracted from three different snap-frozen blocks from MS patients (cases MS114, MS125, and MS163) and six controls (cases C12–101, C12–112, C13–010, C13–022, C14–069, and C14–053) and the expression of P2X7R mRNA was examined by RT-qPCR. Cortical protein extracts (15 μg/well) from different tissue blocks (A5C3, A5B3, A3B1, A1A2, A2A1, and A2B2) of MS cases MS114, MS125, and MS163 were analyzed by western blotting for the modulation of P2X7R, with respect to control cases (C12–101, C12–112, C13–010, C13–022, and C14–069), in both detergent-soluble (B) and -insoluble fractions (C) . GAPDH was used for protein normalization. Statistical significance was calculated by Student’s t-test, * p

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

    P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) monocytes and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive cells within freshly isolated peripheral blood mononuclear cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p
    Figure Legend Snippet: P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) monocytes and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive cells within freshly isolated peripheral blood mononuclear cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p

    Techniques Used: In Vitro, Quantitative RT-PCR, Isolation, Flow Cytometry

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    Alomone Labs p2x7 intracellular epitope antibody
    <t>P2X7</t> receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.
    P2x7 Intracellular Epitope Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    P2X7 receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) expression in both active and inactive subpial lesions of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal triple immunofluorescence analysis performed with antibodies for P2X7R [ (A,D) , red], major histocompatibility complex II (MHC II) [ (B,E) , blue], and myelin basic protein (MBP) [ (C,F) , green] on SPMS frontal cortex sections, shows a chronic active subpial lesion (A–C) with abundant glial fibrillary acidic protein (GFAP)/P2X7R-positive signal (A , inset ) , with reactive MHC II-positive monocyets/macrophages/microglia (blue) and with MBP-positive myelin fibers (green). In a chronic inactive lesion (D–F) , an intense GFAP/P2X7R glial scar is shown [ (D) , inset], with decreased MHC II-positive [ (E) , blue] and MBP-positive [ (F) , green] immunoreactivities.

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Expressing, Immunofluorescence

    Monocyte chemoattractant protein-1 (MCP-1) chemokine colocalizes with P2X7 receptor (P2X7R) on astrocytes in secondary progressive multiple sclerosis (SPMS) frontal cortex. Triple immunofluorescence confocal analysis performed on sections of frontal cortex from control (A–D) and SPMS (E–H) with antibodies for P2X7R [ (A,E) , red], MCP-1 [ (B,F) , green], and glial fibrillary acidic protein [ (C,G) , blue] shows colocalization [ (D,H) , white signal] and strong up-regulation of signals in white matter of SPMS (E–H) .

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: Monocyte chemoattractant protein-1 (MCP-1) chemokine colocalizes with P2X7 receptor (P2X7R) on astrocytes in secondary progressive multiple sclerosis (SPMS) frontal cortex. Triple immunofluorescence confocal analysis performed on sections of frontal cortex from control (A–D) and SPMS (E–H) with antibodies for P2X7R [ (A,E) , red], MCP-1 [ (B,F) , green], and glial fibrillary acidic protein [ (C,G) , blue] shows colocalization [ (D,H) , white signal] and strong up-regulation of signals in white matter of SPMS (E–H) .

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Immunofluorescence

    Spatiotemporal profile of P2X7 receptor (P2X7R) expression in multiple sclerosis (MS). The cartoon describes that P2X7R is down-regulated in monocytes during their activation and extravasation from blood vessel to MS cerebral cortex, while the receptor and the monocyte chemoattractant protein-1 chemokine are up-regulated on MS astrocytes concurrently with increased glial fibrillary acidic protein signal and glial scar formation.

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: Spatiotemporal profile of P2X7 receptor (P2X7R) expression in multiple sclerosis (MS). The cartoon describes that P2X7R is down-regulated in monocytes during their activation and extravasation from blood vessel to MS cerebral cortex, while the receptor and the monocyte chemoattractant protein-1 chemokine are up-regulated on MS astrocytes concurrently with increased glial fibrillary acidic protein signal and glial scar formation.

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Expressing, Activation Assay

    P2X7 receptor (P2X7R) is present on monocytes in blood vessels of secondary progressive multiple sclerosis (SPMS) frontal cortex. (A) Immunohistochemistry on sections from human frontal cortex shows roundish P2X7R-positive cells distributed within distinct clusters throughout the entire SPMS tissue. Confocal double immunofluorescence analysis of these clusters exhibits colocalization of P2X7R protein (red) with CD45 leukocyte marker [ (B) , green]. Staining with Lectin from Lycopersicon esculentum for vascular endothelial cells [ (C,D) , green] clearly shows the presence of P2X7R-positive roundish cells (red) within the lumen of blood vessels (asterisk). Double immunofluorescence of P2X7R-positive clusters shows colocalization of P2X7R (red) with cluster of differentiation 14 (CD14) monocyte/macrophage marker [ (E) , green]. Confocal triple immunofluorescence analysis is then performed with antibodies for CD14 [ (F,G) , green], P2X7R [ (F,G) , red], and microglia/macrophages markers CD68 (F) or major histocompatibility complex II [ (G) , blue]. The asterisk always indicates the lumen of blood vessels, arrows indicate P2X7R-negative cells, and arrowheads P2X7R-positive cells.

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) is present on monocytes in blood vessels of secondary progressive multiple sclerosis (SPMS) frontal cortex. (A) Immunohistochemistry on sections from human frontal cortex shows roundish P2X7R-positive cells distributed within distinct clusters throughout the entire SPMS tissue. Confocal double immunofluorescence analysis of these clusters exhibits colocalization of P2X7R protein (red) with CD45 leukocyte marker [ (B) , green]. Staining with Lectin from Lycopersicon esculentum for vascular endothelial cells [ (C,D) , green] clearly shows the presence of P2X7R-positive roundish cells (red) within the lumen of blood vessels (asterisk). Double immunofluorescence of P2X7R-positive clusters shows colocalization of P2X7R (red) with cluster of differentiation 14 (CD14) monocyte/macrophage marker [ (E) , green]. Confocal triple immunofluorescence analysis is then performed with antibodies for CD14 [ (F,G) , green], P2X7R [ (F,G) , red], and microglia/macrophages markers CD68 (F) or major histocompatibility complex II [ (G) , blue]. The asterisk always indicates the lumen of blood vessels, arrows indicate P2X7R-negative cells, and arrowheads P2X7R-positive cells.

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Immunohistochemistry, Immunofluorescence, Marker, Staining

    P2X7 receptor (P2X7R) is present on astrocytes in the parenchyma of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal analysis of SPMS frontal cortex parenchyma shows absence of colocalization of P2X7R (red) with P2Y12R [ (A) , green], major histocompatibility complex II (MHC II) [ (B) , blue], and myelin basic protein [ (C) , blue], but the presence of merged P2X7R/glial fibrillary acidic protein signals (D–F) . P2X7R/MHC II-positive signal is also visible but confined in the lumen of a blood vessel [ (B) , arrow, pink]. Immunohistochemistry analysis of cortical parenchyma reveals the abundant presence of P2X7R in GM on interlaminar (G) and protoplasmic astrocytes (E) , and in white matter on fibrous astrocytes (I,J) . In (J) , astrocytic “vascular feet” are visible adjacent to the blood vessel walls (arrows).

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) is present on astrocytes in the parenchyma of secondary progressive multiple sclerosis (SPMS) frontal cortex. Confocal analysis of SPMS frontal cortex parenchyma shows absence of colocalization of P2X7R (red) with P2Y12R [ (A) , green], major histocompatibility complex II (MHC II) [ (B) , blue], and myelin basic protein [ (C) , blue], but the presence of merged P2X7R/glial fibrillary acidic protein signals (D–F) . P2X7R/MHC II-positive signal is also visible but confined in the lumen of a blood vessel [ (B) , arrow, pink]. Immunohistochemistry analysis of cortical parenchyma reveals the abundant presence of P2X7R in GM on interlaminar (G) and protoplasmic astrocytes (E) , and in white matter on fibrous astrocytes (I,J) . In (J) , astrocytic “vascular feet” are visible adjacent to the blood vessel walls (arrows).

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Immunohistochemistry

    P2X7 receptor (P2X7R) immunoreactivity is increased with astrogliosis in white matter (WM) of secondary progressive multiple sclerosis (SPMS) frontal cortex parenchyma. Immunohistochemistry analysis of two adjacent WM fields from SPMS frontal cortex characterized, respectively, by hypertrophic astrocytes (B) and glial scar (C) reveals a noteworthy increase in P2X7R-positive astrocytes (B,C) , compared with control case (A) .

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) immunoreactivity is increased with astrogliosis in white matter (WM) of secondary progressive multiple sclerosis (SPMS) frontal cortex parenchyma. Immunohistochemistry analysis of two adjacent WM fields from SPMS frontal cortex characterized, respectively, by hypertrophic astrocytes (B) and glial scar (C) reveals a noteworthy increase in P2X7R-positive astrocytes (B,C) , compared with control case (A) .

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Immunohistochemistry

    P2X7 receptor (P2X7R) mRNA and protein are augmented in secondary progressive multiple sclerosis. (A) Total RNA was extracted from three different snap-frozen blocks from MS patients (cases MS114, MS125, and MS163) and six controls (cases C12–101, C12–112, C13–010, C13–022, C14–069, and C14–053) and the expression of P2X7R mRNA was examined by RT-qPCR. Cortical protein extracts (15 μg/well) from different tissue blocks (A5C3, A5B3, A3B1, A1A2, A2A1, and A2B2) of MS cases MS114, MS125, and MS163 were analyzed by western blotting for the modulation of P2X7R, with respect to control cases (C12–101, C12–112, C13–010, C13–022, and C14–069), in both detergent-soluble (B) and -insoluble fractions (C) . GAPDH was used for protein normalization. Statistical significance was calculated by Student’s t-test, * p

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) mRNA and protein are augmented in secondary progressive multiple sclerosis. (A) Total RNA was extracted from three different snap-frozen blocks from MS patients (cases MS114, MS125, and MS163) and six controls (cases C12–101, C12–112, C13–010, C13–022, C14–069, and C14–053) and the expression of P2X7R mRNA was examined by RT-qPCR. Cortical protein extracts (15 μg/well) from different tissue blocks (A5C3, A5B3, A3B1, A1A2, A2A1, and A2B2) of MS cases MS114, MS125, and MS163 were analyzed by western blotting for the modulation of P2X7R, with respect to control cases (C12–101, C12–112, C13–010, C13–022, and C14–069), in both detergent-soluble (B) and -insoluble fractions (C) . GAPDH was used for protein normalization. Statistical significance was calculated by Student’s t-test, * p

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: Expressing, Quantitative RT-PCR, Western Blot

    P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) monocytes and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive cells within freshly isolated peripheral blood mononuclear cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p

    Journal: Frontiers in Immunology

    Article Title: Modulation of P2X7 Receptor during Inflammation in Multiple Sclerosis

    doi: 10.3389/fimmu.2017.01529

    Figure Lengend Snippet: P2X7 receptor (P2X7R) is down-regulated on circulating multiple sclerosis (MS) monocytes and on healthy donors (HD) monocytes after in vitro induced inflammation. (A) RT-qPCR analysis of P2X7R was performed with freshly isolated monocytes from MS stable ( n = 8), acute patients ( n = 8), and HD ( n = 8). GAPDH was used for normalization. (B) Flow cytometry analysis was used to isolate cluster of differentiation 14 (CD14)-positive monocytes and P2X7R-CD14 double-positive cells within freshly isolated peripheral blood mononuclear cells from acute, stable MS patients, and HD. Cumulative data of P2X7R-positive cells within monocytes are reported as % mean ± SEM ( n = 5). Statistical significance was calculated by ANOVA-Student’s t-test, **** p

    Article Snippet: Proteins were separated by electrophoresis on 10% SDS-PAGE, transferred to nitrocellulose membranes, and processed for western blotting using P2X7-intracellular epitope antibody [1:500, peptide (C)KIRKEFPKTQGQYSGFKYPY, corresponding to amino acids 576–595 of rat P2X7 receptor, Alomone Labs].

    Techniques: In Vitro, Quantitative RT-PCR, Isolation, Flow Cytometry

    P2X7R expression on lymphocytes ( a ), CD4 + ( b ) and CD4 − cells ( c ), and monocytes ( d ) is elevated in patients with neuropathic pain. PBMCs were stained with PerCP-labeled antihuman CD4 antibody and FITC-labeled antihuman P2X7R antibody and analyzed by FACS. The results show a significantly elevated P2X7R expression only in patients suffering from neuropathic pain on the analyzed cell type (lymphocytes ( p = 0.009), CD4 + cells ( p

    Journal: Journal of Neuroinflammation

    Article Title: Differential expression of P2X7 receptor and IL-1β in nociceptive and neuropathic pain

    doi: 10.1186/s12974-016-0565-z

    Figure Lengend Snippet: P2X7R expression on lymphocytes ( a ), CD4 + ( b ) and CD4 − cells ( c ), and monocytes ( d ) is elevated in patients with neuropathic pain. PBMCs were stained with PerCP-labeled antihuman CD4 antibody and FITC-labeled antihuman P2X7R antibody and analyzed by FACS. The results show a significantly elevated P2X7R expression only in patients suffering from neuropathic pain on the analyzed cell type (lymphocytes ( p = 0.009), CD4 + cells ( p

    Article Snippet: For extracellular staining, cells were co-incubated with PerCP-labeled antihuman CD4 antibody (1:50, Biolegend, San Diego, CA, USA) and FITC-labeled antihuman P2X7R antibody (1:100, Alomone Labs, Jerusalem, Israel) at room temperature for 1 hour.

    Techniques: Expressing, Staining, Labeling, FACS

    IL-1β levels are elevated in patients with neuropathic pain. As IL-1β was shown to be the key mediator in P2X7R-pain interplay, we analyzed serum levels of this pro-inflammatory and pro-nociceptive cytokine by multiplex immune assay. Affirmatively to the results with elevated P2X7Rs, we solely found significant increased serum levels in the peripheral blood of patients with NeP * p

    Journal: Journal of Neuroinflammation

    Article Title: Differential expression of P2X7 receptor and IL-1β in nociceptive and neuropathic pain

    doi: 10.1186/s12974-016-0565-z

    Figure Lengend Snippet: IL-1β levels are elevated in patients with neuropathic pain. As IL-1β was shown to be the key mediator in P2X7R-pain interplay, we analyzed serum levels of this pro-inflammatory and pro-nociceptive cytokine by multiplex immune assay. Affirmatively to the results with elevated P2X7Rs, we solely found significant increased serum levels in the peripheral blood of patients with NeP * p

    Article Snippet: For extracellular staining, cells were co-incubated with PerCP-labeled antihuman CD4 antibody (1:50, Biolegend, San Diego, CA, USA) and FITC-labeled antihuman P2X7R antibody (1:100, Alomone Labs, Jerusalem, Israel) at room temperature for 1 hour.

    Techniques: Multiplex Assay

    Exemplary illustration of P2X7 receptor expression. For quantitative analysis of the proportion of CD4 + cells ( a ) and P2X7R protein expression ( b ), cells were stained with PerCP-labeled antihuman CD4 antibody and FITC-labeled antihuman P2X7R antibody. We separately analyzed each subgroup by the mean fluorescence intensity (MFI) of P2X7R expression. Overlay histograms of representative results of P2X7R expression of one patient/healthy volunteer are displayed in histograms

    Journal: Journal of Neuroinflammation

    Article Title: Differential expression of P2X7 receptor and IL-1β in nociceptive and neuropathic pain

    doi: 10.1186/s12974-016-0565-z

    Figure Lengend Snippet: Exemplary illustration of P2X7 receptor expression. For quantitative analysis of the proportion of CD4 + cells ( a ) and P2X7R protein expression ( b ), cells were stained with PerCP-labeled antihuman CD4 antibody and FITC-labeled antihuman P2X7R antibody. We separately analyzed each subgroup by the mean fluorescence intensity (MFI) of P2X7R expression. Overlay histograms of representative results of P2X7R expression of one patient/healthy volunteer are displayed in histograms

    Article Snippet: For extracellular staining, cells were co-incubated with PerCP-labeled antihuman CD4 antibody (1:50, Biolegend, San Diego, CA, USA) and FITC-labeled antihuman P2X7R antibody (1:100, Alomone Labs, Jerusalem, Israel) at room temperature for 1 hour.

    Techniques: Expressing, Staining, Labeling, Fluorescence

    P2RX7 mRNA expression is exclusively increased in patients with neuropathic pain. To confirm the flow cytometric observations with elevated P2X7R protein expression, predominantly on CD4 + cells and only in NeP, we determined the relative P2RX7 mRNA expression of CD4 + cells by qPCR. Affirmatively, increased mRNA levels were consistent with the flow cytometric analyses and exclusively elevated in patients with NeP. ( p = 0.038). * p

    Journal: Journal of Neuroinflammation

    Article Title: Differential expression of P2X7 receptor and IL-1β in nociceptive and neuropathic pain

    doi: 10.1186/s12974-016-0565-z

    Figure Lengend Snippet: P2RX7 mRNA expression is exclusively increased in patients with neuropathic pain. To confirm the flow cytometric observations with elevated P2X7R protein expression, predominantly on CD4 + cells and only in NeP, we determined the relative P2RX7 mRNA expression of CD4 + cells by qPCR. Affirmatively, increased mRNA levels were consistent with the flow cytometric analyses and exclusively elevated in patients with NeP. ( p = 0.038). * p

    Article Snippet: For extracellular staining, cells were co-incubated with PerCP-labeled antihuman CD4 antibody (1:50, Biolegend, San Diego, CA, USA) and FITC-labeled antihuman P2X7R antibody (1:100, Alomone Labs, Jerusalem, Israel) at room temperature for 1 hour.

    Techniques: Expressing, Real-time Polymerase Chain Reaction

    Differential blood count and quantification of CD4 + cells. In order to avoid misinterpretation of potentially elevated P2X7R protein expression based on different cell counts, we quantified numbers of polymorphonuclear leukocytes ( a ), lymphocytes ( b ), and CD4 + cells ( c ). No differences were found between patients suffering from CLBP, NeP, and healthy volunteers. Data are presented as mean ± SD, n = 19, * p

    Journal: Journal of Neuroinflammation

    Article Title: Differential expression of P2X7 receptor and IL-1β in nociceptive and neuropathic pain

    doi: 10.1186/s12974-016-0565-z

    Figure Lengend Snippet: Differential blood count and quantification of CD4 + cells. In order to avoid misinterpretation of potentially elevated P2X7R protein expression based on different cell counts, we quantified numbers of polymorphonuclear leukocytes ( a ), lymphocytes ( b ), and CD4 + cells ( c ). No differences were found between patients suffering from CLBP, NeP, and healthy volunteers. Data are presented as mean ± SD, n = 19, * p

    Article Snippet: For extracellular staining, cells were co-incubated with PerCP-labeled antihuman CD4 antibody (1:50, Biolegend, San Diego, CA, USA) and FITC-labeled antihuman P2X7R antibody (1:100, Alomone Labs, Jerusalem, Israel) at room temperature for 1 hour.

    Techniques: Expressing

    Activation of microglia during the course of EAE is suppressed by the P2X7R antagonist. Immunofluorescence image of Iba-1immunoreactive cells in forebrain of control ( a ), EAE rats ( b , c ) and EAE rats treated with BBG ( d , e ) in asymptomatic (4 d.p.i.) and symptomatic (12 d.p.i.) phases. Insets present magnification of resting ( a ′, e ′) and activated ( c ′) microglial cells. The graph indicates the mean intensity of the fluorescence signal. *** p

    Journal: Inflammopharmacology

    Article Title: Early P2X7R-dependent activation of microglia during the asymptomatic phase of autoimmune encephalomyelitis

    doi: 10.1007/s10787-018-0528-3

    Figure Lengend Snippet: Activation of microglia during the course of EAE is suppressed by the P2X7R antagonist. Immunofluorescence image of Iba-1immunoreactive cells in forebrain of control ( a ), EAE rats ( b , c ) and EAE rats treated with BBG ( d , e ) in asymptomatic (4 d.p.i.) and symptomatic (12 d.p.i.) phases. Insets present magnification of resting ( a ′, e ′) and activated ( c ′) microglial cells. The graph indicates the mean intensity of the fluorescence signal. *** p

    Article Snippet: Immunostaining was performed using primary anti-Iba-1 (1:500; Abcam, Cambridge, GB) and anti-P2X7R antibodies (1:200; Alomone Labs, Jerusalem, Israel), and further with secondary antibody conjugated with Alexa Fluor (1:200; Invitrogen Corp., Carlsbad, CA, USA).

    Techniques: Activation Assay, Immunofluorescence, Fluorescence

    Expression of P2X7R in microglial cells. Immunofluorescence image of double immunostaining Iba-1/P2X7R in forebrain of control ( a ) and EAE rats in asymptomatic (4 d.p.i.) ( b ) and symptomatic (12 d.p.i.) ( c ) phases of the disease. Activated Iba-1/P2X7R-positive cells are seen in immunized rats ( b , c ). Images are representative for each of four animals

    Journal: Inflammopharmacology

    Article Title: Early P2X7R-dependent activation of microglia during the asymptomatic phase of autoimmune encephalomyelitis

    doi: 10.1007/s10787-018-0528-3

    Figure Lengend Snippet: Expression of P2X7R in microglial cells. Immunofluorescence image of double immunostaining Iba-1/P2X7R in forebrain of control ( a ) and EAE rats in asymptomatic (4 d.p.i.) ( b ) and symptomatic (12 d.p.i.) ( c ) phases of the disease. Activated Iba-1/P2X7R-positive cells are seen in immunized rats ( b , c ). Images are representative for each of four animals

    Article Snippet: Immunostaining was performed using primary anti-Iba-1 (1:500; Abcam, Cambridge, GB) and anti-P2X7R antibodies (1:200; Alomone Labs, Jerusalem, Israel), and further with secondary antibody conjugated with Alexa Fluor (1:200; Invitrogen Corp., Carlsbad, CA, USA).

    Techniques: Expressing, Immunofluorescence, Double Immunostaining

    Effects of anti-sense P2X 7 oligonucleotides on BzATP-induced increase in cytosolic calcium (ΔCa 2+ i , A) and on the influx of ethidium bromide (Eth-Br, B) . Cultured mouse wild-type normal keratinocytes were pre-treated with 100 μM anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) for 14 hours followed by 8 hours treatment with 100 μM BzATP. Control – cells treated with the vehicle of the ASO. Levels of ΔCa 2+ i ( A ) and of Eth-Br fluorescence ( B ) were determined as in Fig. 12. The experiments were repeated twice with similar trends.

    Journal: BMC Cancer

    Article Title: Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

    doi: 10.1186/1471-2407-9-114

    Figure Lengend Snippet: Effects of anti-sense P2X 7 oligonucleotides on BzATP-induced increase in cytosolic calcium (ΔCa 2+ i , A) and on the influx of ethidium bromide (Eth-Br, B) . Cultured mouse wild-type normal keratinocytes were pre-treated with 100 μM anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) for 14 hours followed by 8 hours treatment with 100 μM BzATP. Control – cells treated with the vehicle of the ASO. Levels of ΔCa 2+ i ( A ) and of Eth-Br fluorescence ( B ) were determined as in Fig. 12. The experiments were repeated twice with similar trends.

    Article Snippet: Primary antibodies for the immunostaining and Western blots were as follows: Rabbit polyclonal anti-P2X7 receptor antibody, which recognizes the functional full length P2X7 receptor [ ] was from Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDH antibody was from BD Transduction Laboratories (Lexington, KY).

    Techniques: Cell Culture, Allele-specific Oligonucleotide, Fluorescence

    Effects of pre-treatment with anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) (both at 100 μM for 14 hours), and of treatments with BzATP (100 μM, 8 hours) on [ 3 H]thymidine incorporation in mouse wild-type normal keratinocytes (values are means ± SD, n = 4) .

    Journal: BMC Cancer

    Article Title: Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

    doi: 10.1186/1471-2407-9-114

    Figure Lengend Snippet: Effects of pre-treatment with anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) (both at 100 μM for 14 hours), and of treatments with BzATP (100 μM, 8 hours) on [ 3 H]thymidine incorporation in mouse wild-type normal keratinocytes (values are means ± SD, n = 4) .

    Article Snippet: Primary antibodies for the immunostaining and Western blots were as follows: Rabbit polyclonal anti-P2X7 receptor antibody, which recognizes the functional full length P2X7 receptor [ ] was from Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDH antibody was from BD Transduction Laboratories (Lexington, KY).

    Techniques: Allele-specific Oligonucleotide

    Effects of BzATP on apoptosis in cultured mouse keratinocytes . In all experiments levels of apoptosis were normalized to an arbitrary value of 2 in control cells. AU – arbitrary units. A . BzATP dose-response effect in cultured mouse normal (wild-type, C57Bl) keratinocytes (means ± SD, n = 3). Cells were treated with one of the indicated concentrations of BzATP for 8 hours. B . Cultured mouse normal keratinocytes (wild-type, C57Bl) were pre-treated with 100 μM anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) for 14 hours followed by 8 hours treatment with 100 μM BzATP. Control – cells treated with the vehicle of the ASO. Values are means (± SD) of 3 experiments for each condition. Insert in B is Western immunoblot with anti-P2X 7 antibody of lysates of cells treated with ASO or RCO (n = 2). C . BzATP time-response effect in cultured mouse normal keratinocytes (wild-type, C57Bl; filled triangles), or in keratinocytes obtained from P2X 7 -deficient (P2X 7 -/-Pfizer [empty circles] or P2X 7 -/-GSK [filled circles]) mice (both in the C57Bl background). Values are means (± SD) of 3 experiments for each condition. Changes in apoptosis in A and B were determined in terms of solubilized DNA; changes in apoptosis in C were determined using cell-death detection ELISA.

    Journal: BMC Cancer

    Article Title: Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

    doi: 10.1186/1471-2407-9-114

    Figure Lengend Snippet: Effects of BzATP on apoptosis in cultured mouse keratinocytes . In all experiments levels of apoptosis were normalized to an arbitrary value of 2 in control cells. AU – arbitrary units. A . BzATP dose-response effect in cultured mouse normal (wild-type, C57Bl) keratinocytes (means ± SD, n = 3). Cells were treated with one of the indicated concentrations of BzATP for 8 hours. B . Cultured mouse normal keratinocytes (wild-type, C57Bl) were pre-treated with 100 μM anti-sense P2X 7 oligonucleotides (ASO) or random-control P2X 7 oligonucleotides (RCO) for 14 hours followed by 8 hours treatment with 100 μM BzATP. Control – cells treated with the vehicle of the ASO. Values are means (± SD) of 3 experiments for each condition. Insert in B is Western immunoblot with anti-P2X 7 antibody of lysates of cells treated with ASO or RCO (n = 2). C . BzATP time-response effect in cultured mouse normal keratinocytes (wild-type, C57Bl; filled triangles), or in keratinocytes obtained from P2X 7 -deficient (P2X 7 -/-Pfizer [empty circles] or P2X 7 -/-GSK [filled circles]) mice (both in the C57Bl background). Values are means (± SD) of 3 experiments for each condition. Changes in apoptosis in A and B were determined in terms of solubilized DNA; changes in apoptosis in C were determined using cell-death detection ELISA.

    Article Snippet: Primary antibodies for the immunostaining and Western blots were as follows: Rabbit polyclonal anti-P2X7 receptor antibody, which recognizes the functional full length P2X7 receptor [ ] was from Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDH antibody was from BD Transduction Laboratories (Lexington, KY).

    Techniques: Cell Culture, Allele-specific Oligonucleotide, Western Blot, Mouse Assay, Enzyme-linked Immunosorbent Assay

    P2X 7 immunoreactivity in mouse normal skin (A), papilloma (C), and skin cancer tissues (E) (Experiment-1); B, D, F are parallel cross sections, respectively, stained by H E . G . Analysis of P2X 7 immunoreactivity compared among paired histologically normal and cancerous tissues. Bars are means (± SD) of levels in tissues of five mice. H . P2X 7 protein assays in mouse normal and cancer skin tissues. Lysates fractionated by gel electrophoresis were immunoblotted with the anti P2X 7 antibody and membranes were reprobed with the anti GAPDH antibody. Insert in H shows Western immunoblot of lysates of histologically normal and cancerous tissues obtained from the same animal. Similar results were obtained in tissues of two additional mice. Bars show means (± SD) of densitometry results of the P2X 7 -specific 75 KDa bands in tissues of three mice. I . P2X 7 mRNA levels (relative to GAPDH mRNA) (means ± SD) in histologically normal and cancerous tissues obtained from three animals. In G-I data in the normal tissues were normalized in each case to an arbitrary value of 10. * – p

    Journal: BMC Cancer

    Article Title: Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

    doi: 10.1186/1471-2407-9-114

    Figure Lengend Snippet: P2X 7 immunoreactivity in mouse normal skin (A), papilloma (C), and skin cancer tissues (E) (Experiment-1); B, D, F are parallel cross sections, respectively, stained by H E . G . Analysis of P2X 7 immunoreactivity compared among paired histologically normal and cancerous tissues. Bars are means (± SD) of levels in tissues of five mice. H . P2X 7 protein assays in mouse normal and cancer skin tissues. Lysates fractionated by gel electrophoresis were immunoblotted with the anti P2X 7 antibody and membranes were reprobed with the anti GAPDH antibody. Insert in H shows Western immunoblot of lysates of histologically normal and cancerous tissues obtained from the same animal. Similar results were obtained in tissues of two additional mice. Bars show means (± SD) of densitometry results of the P2X 7 -specific 75 KDa bands in tissues of three mice. I . P2X 7 mRNA levels (relative to GAPDH mRNA) (means ± SD) in histologically normal and cancerous tissues obtained from three animals. In G-I data in the normal tissues were normalized in each case to an arbitrary value of 10. * – p

    Article Snippet: Primary antibodies for the immunostaining and Western blots were as follows: Rabbit polyclonal anti-P2X7 receptor antibody, which recognizes the functional full length P2X7 receptor [ ] was from Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDH antibody was from BD Transduction Laboratories (Lexington, KY).

    Techniques: Staining, Mouse Assay, Nucleic Acid Electrophoresis, Western Blot

    Effects in mice in-vivo of local treatment with BzATP on skin apoptosis (Experiment-3) . Animals (n = 5) were treated for 16 weeks either with BzATP, applied locally twice a week on the shaved dorsal skin ( A, B ) or with the vehicle (Control, n = 5, C, D ). At the end of the experiment animals were euthanized and strips were obtained from each animal dorsal skin areas for H E ( B, D ) and P2X 7 /TUNEL co-staining ( E-L ). Arrows in J show increased TUNEL staining co-localizing with P2X 7 immunoreactivity in epidermal cells of the basal/parabasal layers (horizontal arrow) and of epidermal hair shaft cells (vertical arrow). Assays were repeated 3–5 times with similar trends.

    Journal: BMC Cancer

    Article Title: Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

    doi: 10.1186/1471-2407-9-114

    Figure Lengend Snippet: Effects in mice in-vivo of local treatment with BzATP on skin apoptosis (Experiment-3) . Animals (n = 5) were treated for 16 weeks either with BzATP, applied locally twice a week on the shaved dorsal skin ( A, B ) or with the vehicle (Control, n = 5, C, D ). At the end of the experiment animals were euthanized and strips were obtained from each animal dorsal skin areas for H E ( B, D ) and P2X 7 /TUNEL co-staining ( E-L ). Arrows in J show increased TUNEL staining co-localizing with P2X 7 immunoreactivity in epidermal cells of the basal/parabasal layers (horizontal arrow) and of epidermal hair shaft cells (vertical arrow). Assays were repeated 3–5 times with similar trends.

    Article Snippet: Primary antibodies for the immunostaining and Western blots were as follows: Rabbit polyclonal anti-P2X7 receptor antibody, which recognizes the functional full length P2X7 receptor [ ] was from Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDH antibody was from BD Transduction Laboratories (Lexington, KY).

    Techniques: Mouse Assay, In Vivo, TUNEL Assay, Staining

    Effects of treatments with BzATP on P2X 7 expression and apoptosis in DMBA/TPA – induced skin papillomas (A-D) and cancers (E-H) (Experiment-1) . Assays were repeated 4 times with similar trends. C, D, G, H are parallel cross sections to A, B, E, F , respectively.

    Journal: BMC Cancer

    Article Title: Activation of P2X7-mediated apoptosis Inhibits DMBA/TPA-induced formation of skin papillomas and cancer in mice

    doi: 10.1186/1471-2407-9-114

    Figure Lengend Snippet: Effects of treatments with BzATP on P2X 7 expression and apoptosis in DMBA/TPA – induced skin papillomas (A-D) and cancers (E-H) (Experiment-1) . Assays were repeated 4 times with similar trends. C, D, G, H are parallel cross sections to A, B, E, F , respectively.

    Article Snippet: Primary antibodies for the immunostaining and Western blots were as follows: Rabbit polyclonal anti-P2X7 receptor antibody, which recognizes the functional full length P2X7 receptor [ ] was from Alomone Laboratories (Jerusalem, Israel); rabbit anti-GAPDH antibody was from BD Transduction Laboratories (Lexington, KY).

    Techniques: Expressing