p2x7 receptors  (Alomone Labs)


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    Alomone Labs p2x7 receptors
    Inhibition of <t>P2X7</t> receptors attenuated surgery-induced increase of P2X7 receptor and caspase 1 expression. Hippocampus was harvested 6 h or 7 days after the surgery under isoflurane anesthesia. A and B: representative images of Western blotting. C: quantitative data of P2X7 receptors. D: quantitative data of P20. E: quantitative data of precursor caspase 1. Results are mean ± S.E.M. (n = 6). * P
    P2x7 Receptors, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Images

    1) Product Images from "Critical role of P2X7 receptors in the neuroinflammation and cognitive dysfunction after surgery"

    Article Title: Critical role of P2X7 receptors in the neuroinflammation and cognitive dysfunction after surgery

    Journal: Brain, behavior, and immunity

    doi: 10.1016/j.bbi.2017.01.005

    Inhibition of P2X7 receptors attenuated surgery-induced increase of P2X7 receptor and caspase 1 expression. Hippocampus was harvested 6 h or 7 days after the surgery under isoflurane anesthesia. A and B: representative images of Western blotting. C: quantitative data of P2X7 receptors. D: quantitative data of P20. E: quantitative data of precursor caspase 1. Results are mean ± S.E.M. (n = 6). * P
    Figure Legend Snippet: Inhibition of P2X7 receptors attenuated surgery-induced increase of P2X7 receptor and caspase 1 expression. Hippocampus was harvested 6 h or 7 days after the surgery under isoflurane anesthesia. A and B: representative images of Western blotting. C: quantitative data of P2X7 receptors. D: quantitative data of P20. E: quantitative data of precursor caspase 1. Results are mean ± S.E.M. (n = 6). * P

    Techniques Used: Inhibition, Expressing, Western Blot

    Surgery increased P2X7 receptor and caspase 1 expression. Hippocampus was harvested 6 h after the surgery for immunohistochemical staining or 24 h after the surgery for Western blotting. A: representative images of immunostaining. Scale bar = 100 µm. B: representative images of Western blotting. C: quantitative data of Western blotting. Results are mean ± S.E.M. (n = 5 – 6). * P
    Figure Legend Snippet: Surgery increased P2X7 receptor and caspase 1 expression. Hippocampus was harvested 6 h after the surgery for immunohistochemical staining or 24 h after the surgery for Western blotting. A: representative images of immunostaining. Scale bar = 100 µm. B: representative images of Western blotting. C: quantitative data of Western blotting. Results are mean ± S.E.M. (n = 5 – 6). * P

    Techniques Used: Expressing, Immunohistochemistry, Staining, Western Blot, Immunostaining

    Inhibition of P2X7 receptors attenuated surgery-induced learning and memory impairment. Mice were started to be tested by Barnes maze and fear conditioning 2 weeks after the surgery under isoflurane anesthesia. A: training sessions of Barnes maze. Results are mean ± S.E.M. (n = 12). * P
    Figure Legend Snippet: Inhibition of P2X7 receptors attenuated surgery-induced learning and memory impairment. Mice were started to be tested by Barnes maze and fear conditioning 2 weeks after the surgery under isoflurane anesthesia. A: training sessions of Barnes maze. Results are mean ± S.E.M. (n = 12). * P

    Techniques Used: Inhibition, Mouse Assay

    Inhibition of P2X7 receptors attenuated surgery-induced neuroinflammation. Hippocampus was harvested 6 h or 7 days after the surgery. A: representative images of Iba-1 staining of hippocampus harvested 6 h after the surgery. Scale bar = 100 µm. B: quantitative data of Iba-1 of hippocampus harvested 6 h after the surgery. C: representative images of Iba-1 staining of hippocampus harvested 7 days after the surgery. D: quantitative data of Iba-1 of hippocampus harvested 7 days after the surgery. Results are mean ± S.E.M. (n = 5 for panels B and D). * P
    Figure Legend Snippet: Inhibition of P2X7 receptors attenuated surgery-induced neuroinflammation. Hippocampus was harvested 6 h or 7 days after the surgery. A: representative images of Iba-1 staining of hippocampus harvested 6 h after the surgery. Scale bar = 100 µm. B: quantitative data of Iba-1 of hippocampus harvested 6 h after the surgery. C: representative images of Iba-1 staining of hippocampus harvested 7 days after the surgery. D: quantitative data of Iba-1 of hippocampus harvested 7 days after the surgery. Results are mean ± S.E.M. (n = 5 for panels B and D). * P

    Techniques Used: Inhibition, Staining

    2) Product Images from "Critical role of P2X7 receptors in the neuroinflammation and cognitive dysfunction after surgery"

    Article Title: Critical role of P2X7 receptors in the neuroinflammation and cognitive dysfunction after surgery

    Journal: Brain, behavior, and immunity

    doi: 10.1016/j.bbi.2017.01.005

    Inhibition of P2X7 receptors attenuated surgery-induced increase of P2X7 receptor and caspase 1 expression. Hippocampus was harvested 6 h or 7 days after the surgery under isoflurane anesthesia. A and B: representative images of Western blotting. C: quantitative data of P2X7 receptors. D: quantitative data of P20. E: quantitative data of precursor caspase 1. Results are mean ± S.E.M. (n = 6). * P
    Figure Legend Snippet: Inhibition of P2X7 receptors attenuated surgery-induced increase of P2X7 receptor and caspase 1 expression. Hippocampus was harvested 6 h or 7 days after the surgery under isoflurane anesthesia. A and B: representative images of Western blotting. C: quantitative data of P2X7 receptors. D: quantitative data of P20. E: quantitative data of precursor caspase 1. Results are mean ± S.E.M. (n = 6). * P

    Techniques Used: Inhibition, Expressing, Western Blot

    Surgery increased P2X7 receptor and caspase 1 expression. Hippocampus was harvested 6 h after the surgery for immunohistochemical staining or 24 h after the surgery for Western blotting. A: representative images of immunostaining. Scale bar = 100 µm. B: representative images of Western blotting. C: quantitative data of Western blotting. Results are mean ± S.E.M. (n = 5 – 6). * P
    Figure Legend Snippet: Surgery increased P2X7 receptor and caspase 1 expression. Hippocampus was harvested 6 h after the surgery for immunohistochemical staining or 24 h after the surgery for Western blotting. A: representative images of immunostaining. Scale bar = 100 µm. B: representative images of Western blotting. C: quantitative data of Western blotting. Results are mean ± S.E.M. (n = 5 – 6). * P

    Techniques Used: Expressing, Immunohistochemistry, Staining, Western Blot, Immunostaining

    Inhibition of P2X7 receptors attenuated surgery-induced learning and memory impairment. Mice were started to be tested by Barnes maze and fear conditioning 2 weeks after the surgery under isoflurane anesthesia. A: training sessions of Barnes maze. Results are mean ± S.E.M. (n = 12). * P
    Figure Legend Snippet: Inhibition of P2X7 receptors attenuated surgery-induced learning and memory impairment. Mice were started to be tested by Barnes maze and fear conditioning 2 weeks after the surgery under isoflurane anesthesia. A: training sessions of Barnes maze. Results are mean ± S.E.M. (n = 12). * P

    Techniques Used: Inhibition, Mouse Assay

    Inhibition of P2X7 receptors attenuated surgery-induced neuroinflammation. Hippocampus was harvested 6 h or 7 days after the surgery. A: representative images of Iba-1 staining of hippocampus harvested 6 h after the surgery. Scale bar = 100 µm. B: quantitative data of Iba-1 of hippocampus harvested 6 h after the surgery. C: representative images of Iba-1 staining of hippocampus harvested 7 days after the surgery. D: quantitative data of Iba-1 of hippocampus harvested 7 days after the surgery. Results are mean ± S.E.M. (n = 5 for panels B and D). * P
    Figure Legend Snippet: Inhibition of P2X7 receptors attenuated surgery-induced neuroinflammation. Hippocampus was harvested 6 h or 7 days after the surgery. A: representative images of Iba-1 staining of hippocampus harvested 6 h after the surgery. Scale bar = 100 µm. B: quantitative data of Iba-1 of hippocampus harvested 6 h after the surgery. C: representative images of Iba-1 staining of hippocampus harvested 7 days after the surgery. D: quantitative data of Iba-1 of hippocampus harvested 7 days after the surgery. Results are mean ± S.E.M. (n = 5 for panels B and D). * P

    Techniques Used: Inhibition, Staining

    3) Product Images from "5-Nitro-2-(3-phenylpropylamino)benzoic Acid (NPPB) Stimulates Cellular ATP Release through Exocytosis of ATP-enriched Vesicles *"

    Article Title: 5-Nitro-2-(3-phenylpropylamino)benzoic Acid (NPPB) Stimulates Cellular ATP Release through Exocytosis of ATP-enriched Vesicles *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M109.046193

    NPPB stimulates ATP release in HEK293 cells. A , HEK293 cells were incubated with a plasmid containing an EGFP vector and a rat P2X7 receptor ( bottom left panel ). These cells exhibit evenly distributed fluorescence as indicative of the P2X7 receptor expression.
    Figure Legend Snippet: NPPB stimulates ATP release in HEK293 cells. A , HEK293 cells were incubated with a plasmid containing an EGFP vector and a rat P2X7 receptor ( bottom left panel ). These cells exhibit evenly distributed fluorescence as indicative of the P2X7 receptor expression.

    Techniques Used: Incubation, Plasmid Preparation, Fluorescence, Expressing

    The effects of NPPB on viability of HEK293 cells. Calcein fluorescence was measured in HEK293 ( open circles ) and HEK293-P2X7 cells ( closed circles ). A , addition of 100 μ m NPPB (at the arrow ) did not significantly change calcein fluorescence in
    Figure Legend Snippet: The effects of NPPB on viability of HEK293 cells. Calcein fluorescence was measured in HEK293 ( open circles ) and HEK293-P2X7 cells ( closed circles ). A , addition of 100 μ m NPPB (at the arrow ) did not significantly change calcein fluorescence in

    Techniques Used: Fluorescence

    NPPB-evoked exocytosis and ATP release. The magnitude of exocytosis (ΔF) is plotted versus relative luminescence for different NPPB concentrations (50 - 200 μ m , closed circles ). The data were pooled from HTC, Mz-Cha-1, HEK293, and HEK293-P2X7
    Figure Legend Snippet: NPPB-evoked exocytosis and ATP release. The magnitude of exocytosis (ΔF) is plotted versus relative luminescence for different NPPB concentrations (50 - 200 μ m , closed circles ). The data were pooled from HTC, Mz-Cha-1, HEK293, and HEK293-P2X7

    Techniques Used:

    Localization of P2X7 receptors. HTC and Mz-Cha-1 cells were fixed and permeabilized, and then incubated with primary P2X7 ectoAb ( left panels ) or control nonspecific isotype IgG antibody ( right panels ). To visualize the cellular distribution of P2X7 receptors,
    Figure Legend Snippet: Localization of P2X7 receptors. HTC and Mz-Cha-1 cells were fixed and permeabilized, and then incubated with primary P2X7 ectoAb ( left panels ) or control nonspecific isotype IgG antibody ( right panels ). To visualize the cellular distribution of P2X7 receptors,

    Techniques Used: Incubation

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    Alomone Labs rabbit polyclonal anti human p2x7 receptor atto 488
    TLR4 signaling influences <t>P2X7</t> receptor distribution. Representative confocal microphotographs showing (A) HuC/D (magenta) and P2X7Rs (yellow) distribution and (B) analysis of P2X7Rs fluorescence intensities in WT and TLR4 -/- preparations ( N = 5 mice/group). Cell nuclei were stained with TOTO-3 (blue). Bars = 22 μm. ∗ P
    Rabbit Polyclonal Anti Human P2x7 Receptor Atto 488, 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
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    95
    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 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Average 95 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    TLR4 signaling influences P2X7 receptor distribution. Representative confocal microphotographs showing (A) HuC/D (magenta) and P2X7Rs (yellow) distribution and (B) analysis of P2X7Rs fluorescence intensities in WT and TLR4 -/- preparations ( N = 5 mice/group). Cell nuclei were stained with TOTO-3 (blue). Bars = 22 μm. ∗ P

    Journal: Frontiers in Pharmacology

    Article Title: Toll-Like Receptor 4 Modulates Small Intestine Neuromuscular Function through Nitrergic and Purinergic Pathways

    doi: 10.3389/fphar.2017.00350

    Figure Lengend Snippet: TLR4 signaling influences P2X7 receptor distribution. Representative confocal microphotographs showing (A) HuC/D (magenta) and P2X7Rs (yellow) distribution and (B) analysis of P2X7Rs fluorescence intensities in WT and TLR4 -/- preparations ( N = 5 mice/group). Cell nuclei were stained with TOTO-3 (blue). Bars = 22 μm. ∗ P

    Article Snippet: LMMP whole mount preparations or ileal cryosections were then incubated overnight at room temperature with the following antibodies: chicken polyclonal anti-mouse glial fibrillary acidic protein (GFAP; 1:100; Abcam, Milan, Italy), rabbit polyclonal anti-human GFAP (1:200; Merck Millipore Corporation, Milan, Italy), mouse biotin-conjugated anti-human HuC/D (1:50; Thermo Fisher Scientific, Milan, Italy), rabbit polyclonal anti-mouse neuronal nitric oxide synthase (nNOS, 1:100; Thermo Fisher Scientific, Milan, Italy), rabbit polyclonal anti-human inducible NOS (iNOS; 1:50, Santa Cruz Biotechnology, Milan, Italy), rabbit polyclonal anti-human vasoactive intestinal peptide (VIP, 1:100; GenWay Biotech, Milan, Italy), rabbit polyclonal anti-human P2X7 receptor-ATTO-488 (1:100; Alomone labs, Jerusalem, Israel), rabbit polyclonal anti-human P2Y1 receptor (1:100; Alomone labs, Jerusalem, Israel), rabbit monoclonal anti-human S100β (1:100; Merck Millipore Corporation, Milan, Italy) and guinea pig polyclonal anti-mouse substance P (1:100; Abcam, Milan, Italy).

    Techniques: Fluorescence, Mouse Assay, Staining

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0155107

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

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

    Techniques: Expressing, Cell Culture

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0155107

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

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

    Techniques: Western Blot, Functional Assay, Variant Assay

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0155107

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

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

    Techniques:

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

    Journal: PLoS ONE

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

    doi: 10.1371/journal.pone.0155107

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

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

    Techniques:

    BzATP-mediated Egr target gene induction is dependent on the P2X7-Egr pathway Parental N9, shEgr/N9 or shP2X7/N9 microglia were treated with either vehicle (250 mM Hepes) or BzATP (150μM) for 1 hour (A, B) or 20 hours (C). (A) BzATP-induced Egr expression is blunted in shEgr/N9 cells. (B) RNAi to P2X7 and Egr factors prevents BzATP-induced Egr target gene (Nab-1/Nab-2) activation. Nab gene induction by BzATP in parental cells ranged from 3-14 fold over vehicle treatment. (C) RNAi to Egr factors, but not to P2X7 receptors, decreases VEGF expression. The data are graphed as fold induction relative to BzATP-treated parental N9 cells for each gene and represent the means of 4-8 independent experiments + SEM.

    Journal: Glia

    Article Title: The P2X7-Egr Pathway Regulates Nucleotide-Dependent Inflammatory Gene Expression in Microglia

    doi: 10.1002/glia.21071

    Figure Lengend Snippet: BzATP-mediated Egr target gene induction is dependent on the P2X7-Egr pathway Parental N9, shEgr/N9 or shP2X7/N9 microglia were treated with either vehicle (250 mM Hepes) or BzATP (150μM) for 1 hour (A, B) or 20 hours (C). (A) BzATP-induced Egr expression is blunted in shEgr/N9 cells. (B) RNAi to P2X7 and Egr factors prevents BzATP-induced Egr target gene (Nab-1/Nab-2) activation. Nab gene induction by BzATP in parental cells ranged from 3-14 fold over vehicle treatment. (C) RNAi to Egr factors, but not to P2X7 receptors, decreases VEGF expression. The data are graphed as fold induction relative to BzATP-treated parental N9 cells for each gene and represent the means of 4-8 independent experiments + SEM.

    Article Snippet: The levels of p44/42, p38, iNOS and P2X7 were ascertained using: anti-active p44/42 antibodies (1:1000, Cell Signaling, Danver, MA), anti-active p38 MAP kinase antibodies (1:1000, Cell Signaling, Danver, MA), anti-iNOS antibodies (1:2000; Transduction Laboratories, Lexington, KY), and anti-P2X7 antibodies (1:200; Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing, Activation Assay

    BzATP abrogates LPS-induced HT22 hippocampal neuron death via P2X7 receptors Parental N9 and shP2X7/N9 cells were treated overnight with vehicle (250 mM Hepes) or LPS (1 μg/mL) in the presence and absence of BzATP (150 μM). Microglial conditioned medium was applied to HT22 neurons for 18-22 hours after which time neuron survival was assessed by the MTS mitochondrial function assay. The data are graphed as % of neurons surviving relative to control treatment and represent the means of 3-6 independent experiments ± SEM. ** p

    Journal: Glia

    Article Title: The P2X7-Egr Pathway Regulates Nucleotide-Dependent Inflammatory Gene Expression in Microglia

    doi: 10.1002/glia.21071

    Figure Lengend Snippet: BzATP abrogates LPS-induced HT22 hippocampal neuron death via P2X7 receptors Parental N9 and shP2X7/N9 cells were treated overnight with vehicle (250 mM Hepes) or LPS (1 μg/mL) in the presence and absence of BzATP (150 μM). Microglial conditioned medium was applied to HT22 neurons for 18-22 hours after which time neuron survival was assessed by the MTS mitochondrial function assay. The data are graphed as % of neurons surviving relative to control treatment and represent the means of 3-6 independent experiments ± SEM. ** p

    Article Snippet: The levels of p44/42, p38, iNOS and P2X7 were ascertained using: anti-active p44/42 antibodies (1:1000, Cell Signaling, Danver, MA), anti-active p38 MAP kinase antibodies (1:1000, Cell Signaling, Danver, MA), anti-iNOS antibodies (1:2000; Transduction Laboratories, Lexington, KY), and anti-P2X7 antibodies (1:200; Alomone Labs, Jerusalem, Israel).

    Techniques: Functional Assay

    BzATP-induced IL-6 expression is blocked in microglia stably expressing short hairpins to P2X7 and Egr factors Parental N9, shP2X7/N9 and shEgr/N9 cells were treated overnight with either vehicle (250 mM Hepes) or BzATP (150 μM). (A) BzATP increases IL-6 expression in parental N9 microglia. (B) BzATP-induced IL-6 expression is strongly attenuated in shP2X7/N9 and shEgr/N9 cells. The data are graphed as fold induction relative to either vehicle (A) or BzATP treatment (B) in each cell type. Treatments represent the mean ± SEM of at least 4 independent experiments. ***p

    Journal: Glia

    Article Title: The P2X7-Egr Pathway Regulates Nucleotide-Dependent Inflammatory Gene Expression in Microglia

    doi: 10.1002/glia.21071

    Figure Lengend Snippet: BzATP-induced IL-6 expression is blocked in microglia stably expressing short hairpins to P2X7 and Egr factors Parental N9, shP2X7/N9 and shEgr/N9 cells were treated overnight with either vehicle (250 mM Hepes) or BzATP (150 μM). (A) BzATP increases IL-6 expression in parental N9 microglia. (B) BzATP-induced IL-6 expression is strongly attenuated in shP2X7/N9 and shEgr/N9 cells. The data are graphed as fold induction relative to either vehicle (A) or BzATP treatment (B) in each cell type. Treatments represent the mean ± SEM of at least 4 independent experiments. ***p

    Article Snippet: The levels of p44/42, p38, iNOS and P2X7 were ascertained using: anti-active p44/42 antibodies (1:1000, Cell Signaling, Danver, MA), anti-active p38 MAP kinase antibodies (1:1000, Cell Signaling, Danver, MA), anti-iNOS antibodies (1:2000; Transduction Laboratories, Lexington, KY), and anti-P2X7 antibodies (1:200; Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing, Stable Transfection

    P2X7 purinergic receptors mediate induction of Egr factors by BzATP (A) P2X7 receptor antagonists significantly attenuate BzATP-induced Egr expression. N9 microglia were pre-treated with vehicle (250 mM Hepes) or the P2X7 receptor antagonists oATP (500μM) for 2 hours, or Brilliant Blue G (1mM) or KN-62 (25μM) for 30 minutes prior to stimulation with either vehicle or BzATP (150 μM) for 1 hour. The data shown represent the mean ± SEM of at least 3 independent experiments, expressed as percent gene induction relative to N9 cells treated with BzATP alone. (B) Cells stably expressing short hairpins to P2X7 receptors (shP2X7/N9) show decreased P2X7 mRNA (upper panel) and protein levels (lower panel). In the representative Western blot shown, a high molecular weight, non-specific band is observed using this P2X7 antibody. (C) YO-PRO uptake stimulated by BzATP in parental N9 cells (upper left panel) is abrogated in shP2X7/N9 (upper right panel), but not in shP2X4/N9 (lower left panel) or shEgr/N9 (lower right panel) cells. (D) BzATP-induced Egr expression is greatly attenuated in shP2X7/N9 but not in shP2X4/N9 cells treated with vehicle or BzATP for 1 hour. **p

    Journal: Glia

    Article Title: The P2X7-Egr Pathway Regulates Nucleotide-Dependent Inflammatory Gene Expression in Microglia

    doi: 10.1002/glia.21071

    Figure Lengend Snippet: P2X7 purinergic receptors mediate induction of Egr factors by BzATP (A) P2X7 receptor antagonists significantly attenuate BzATP-induced Egr expression. N9 microglia were pre-treated with vehicle (250 mM Hepes) or the P2X7 receptor antagonists oATP (500μM) for 2 hours, or Brilliant Blue G (1mM) or KN-62 (25μM) for 30 minutes prior to stimulation with either vehicle or BzATP (150 μM) for 1 hour. The data shown represent the mean ± SEM of at least 3 independent experiments, expressed as percent gene induction relative to N9 cells treated with BzATP alone. (B) Cells stably expressing short hairpins to P2X7 receptors (shP2X7/N9) show decreased P2X7 mRNA (upper panel) and protein levels (lower panel). In the representative Western blot shown, a high molecular weight, non-specific band is observed using this P2X7 antibody. (C) YO-PRO uptake stimulated by BzATP in parental N9 cells (upper left panel) is abrogated in shP2X7/N9 (upper right panel), but not in shP2X4/N9 (lower left panel) or shEgr/N9 (lower right panel) cells. (D) BzATP-induced Egr expression is greatly attenuated in shP2X7/N9 but not in shP2X4/N9 cells treated with vehicle or BzATP for 1 hour. **p

    Article Snippet: The levels of p44/42, p38, iNOS and P2X7 were ascertained using: anti-active p44/42 antibodies (1:1000, Cell Signaling, Danver, MA), anti-active p38 MAP kinase antibodies (1:1000, Cell Signaling, Danver, MA), anti-iNOS antibodies (1:2000; Transduction Laboratories, Lexington, KY), and anti-P2X7 antibodies (1:200; Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing, Stable Transfection, Western Blot, Molecular Weight

    BzATP-induced alterations in LPS-stimulated NO and TNF-α production requires the P2X7-Egr pathway Parental N9, shP2X7/N9 and shEgr/N9 cells were treated with vehicle (250 mM Hepes), LPS (1 μg/mL), or LPS+BzATP (150 μM) overnight. (A) BzATP-mediated inhibition of LPS-stimulated iNOS expression does not occur in shP2X7/N9 and shEgr/N9 cells. (B) BzATP-mediated augmentation of LPS-induced TNF-α expression is abrogated in shP2X7/N9 and shEgr/N9 cells. (C) Expression of TGF-β and β-actin are unchanged in shEgr/N9 cells treated with vehicle or LPS. (D) BzATP treatment increases LPS-stimulated IL-10 mRNA levels in both parental N9 and shEgr/N9 cells. The means ± SEM are graphed as fold induction relative to vehicle treatment (C) or LPS treatment (A, B, D) in 3-8 independent experiments. * p

    Journal: Glia

    Article Title: The P2X7-Egr Pathway Regulates Nucleotide-Dependent Inflammatory Gene Expression in Microglia

    doi: 10.1002/glia.21071

    Figure Lengend Snippet: BzATP-induced alterations in LPS-stimulated NO and TNF-α production requires the P2X7-Egr pathway Parental N9, shP2X7/N9 and shEgr/N9 cells were treated with vehicle (250 mM Hepes), LPS (1 μg/mL), or LPS+BzATP (150 μM) overnight. (A) BzATP-mediated inhibition of LPS-stimulated iNOS expression does not occur in shP2X7/N9 and shEgr/N9 cells. (B) BzATP-mediated augmentation of LPS-induced TNF-α expression is abrogated in shP2X7/N9 and shEgr/N9 cells. (C) Expression of TGF-β and β-actin are unchanged in shEgr/N9 cells treated with vehicle or LPS. (D) BzATP treatment increases LPS-stimulated IL-10 mRNA levels in both parental N9 and shEgr/N9 cells. The means ± SEM are graphed as fold induction relative to vehicle treatment (C) or LPS treatment (A, B, D) in 3-8 independent experiments. * p

    Article Snippet: The levels of p44/42, p38, iNOS and P2X7 were ascertained using: anti-active p44/42 antibodies (1:1000, Cell Signaling, Danver, MA), anti-active p38 MAP kinase antibodies (1:1000, Cell Signaling, Danver, MA), anti-iNOS antibodies (1:2000; Transduction Laboratories, Lexington, KY), and anti-P2X7 antibodies (1:200; Alomone Labs, Jerusalem, Israel).

    Techniques: Inhibition, Expressing

    Enhanced IL-1β secretion in dysferlin-deficient primary mature myotubes. Primary C57Bl/6, SJL/J, and MDX muscle cultures were grown for 7 days then differentiated into mature myotubes for 2 days. Myotubes were stimulated with LPS and BzATP, and then supernatants and lysates were analyzed for ELISA and Western blotting. A: Western blot analysis shows increased expression of ASC-1, pro-caspase-1, pro-IL-1β, and P2X7 in lysates of dysferlin-deficient muscle. B: ELISA shows the effect of LPS and BzATP on IL-1β secretion from C57Bl/6J, SJL/J, and MDX mature myotubes: stimulation with LPS and BzATP induced increased secretion of IL-1β in dysferlin- and dystrophin-deficient primary myotubes compared with C57Bl/6J muscle ( n = 3 ± SEM). C: shRNA knockdown of dysferlin increases LPS/BzATP-stimulated IL-1β secretion. Western blot shows dysferlin expression from nontransfected (NT) primary muscle cells, or cells transiently transfected with scrambled shRNA (Sc) or dysferlin shRNA (Dy). Vinculin was used as a loading control. D: ELISA shows up-regulated IL-1β secretion in primary muscle cells transiently transfected with dysferlin shRNA compared with controls ( left ). Up-regulated IL-1β secretion was also observed in RAW cells that were transiently transfected with dysferlin shRNA compared with controls ( right ).

    Journal: The American Journal of Pathology

    Article Title: Inflammasome Up-Regulation and Activation in Dysferlin-Deficient Skeletal Muscle

    doi: 10.2353/ajpath.2010.090058

    Figure Lengend Snippet: Enhanced IL-1β secretion in dysferlin-deficient primary mature myotubes. Primary C57Bl/6, SJL/J, and MDX muscle cultures were grown for 7 days then differentiated into mature myotubes for 2 days. Myotubes were stimulated with LPS and BzATP, and then supernatants and lysates were analyzed for ELISA and Western blotting. A: Western blot analysis shows increased expression of ASC-1, pro-caspase-1, pro-IL-1β, and P2X7 in lysates of dysferlin-deficient muscle. B: ELISA shows the effect of LPS and BzATP on IL-1β secretion from C57Bl/6J, SJL/J, and MDX mature myotubes: stimulation with LPS and BzATP induced increased secretion of IL-1β in dysferlin- and dystrophin-deficient primary myotubes compared with C57Bl/6J muscle ( n = 3 ± SEM). C: shRNA knockdown of dysferlin increases LPS/BzATP-stimulated IL-1β secretion. Western blot shows dysferlin expression from nontransfected (NT) primary muscle cells, or cells transiently transfected with scrambled shRNA (Sc) or dysferlin shRNA (Dy). Vinculin was used as a loading control. D: ELISA shows up-regulated IL-1β secretion in primary muscle cells transiently transfected with dysferlin shRNA compared with controls ( left ). Up-regulated IL-1β secretion was also observed in RAW cells that were transiently transfected with dysferlin shRNA compared with controls ( right ).

    Article Snippet: Western blotting was performed with antibodies recognizing the P2X7 receptor (Alomone Lab, Israel; 1:500 dilution), NALP-3 (Alexis Biochem, San Diego, CA; 1:1000 dilution), ASC-1 (Alexis Biochem;1:1000 dilution), caspase-1 (Imgenex, San Diego, CA; 1:1000 dilution), IL-1β (a gift from the National Cancer Institute Biometrics Research Branch (NCIBRB) Preclinical Repository, NCI, Fredrick, MD; 1:10,000 dilution), dysferlin (Novocastra Newcastle on Tyne, UK; 1:500 dilution), and Vinculin (Sigma- Aldrich, St. Louis, MO; 1:2500 dilution) and developed by using Enhanced Chemiluminescence (ECL) (GE Health care, Piscataway, NJ).

    Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Expressing, shRNA, Transfection

    Mechanisms of inflammation and muscle fiber damage in dysferlin deficiency. Factors that initiate and perpetuate the disease are not well understood. It is likely that age-related physiological changes in skeletal muscle in conjunction with environmental insults may initiate the disease process in LGMD2B. Because dysferlin is expressed both in skeletal muscle and macrophages, it is likely that the phenotype is due to abnormalities in both cell types. Here we propose that increase in vesicular trafficking and plasma membrane repair defects results in release in ATP and other endogenous danger/alarm signals (eg, HMGB1, S100 proteins, etc). These, in turn, bind to their cellular receptors (TRLs, P2X7 receptors) and activate inflammasome (ASC-1, NALP-3, caspase-1, and IL-1β) and NF-κB pathways. Extracellular ATP and complement components, which are also activated, are known to induce additional pores on plasma membrane resulting in an influx of calcium and activation of calcium-dependent proteases, pyroptosis, and pyronecrosis. Further, activation of NF-κB in this disease may induce not only muscle fiber wasting but also inhibition of myogenesis. All of these downstream processes activate not only inflammation and fibrosis but significant muscle fiber damage and dysfunction. HSP, heat-shock protein; MAC, membrane attack complex; IRAK, IL-1R-associated kinase.

    Journal: The American Journal of Pathology

    Article Title: Inflammasome Up-Regulation and Activation in Dysferlin-Deficient Skeletal Muscle

    doi: 10.2353/ajpath.2010.090058

    Figure Lengend Snippet: Mechanisms of inflammation and muscle fiber damage in dysferlin deficiency. Factors that initiate and perpetuate the disease are not well understood. It is likely that age-related physiological changes in skeletal muscle in conjunction with environmental insults may initiate the disease process in LGMD2B. Because dysferlin is expressed both in skeletal muscle and macrophages, it is likely that the phenotype is due to abnormalities in both cell types. Here we propose that increase in vesicular trafficking and plasma membrane repair defects results in release in ATP and other endogenous danger/alarm signals (eg, HMGB1, S100 proteins, etc). These, in turn, bind to their cellular receptors (TRLs, P2X7 receptors) and activate inflammasome (ASC-1, NALP-3, caspase-1, and IL-1β) and NF-κB pathways. Extracellular ATP and complement components, which are also activated, are known to induce additional pores on plasma membrane resulting in an influx of calcium and activation of calcium-dependent proteases, pyroptosis, and pyronecrosis. Further, activation of NF-κB in this disease may induce not only muscle fiber wasting but also inhibition of myogenesis. All of these downstream processes activate not only inflammation and fibrosis but significant muscle fiber damage and dysfunction. HSP, heat-shock protein; MAC, membrane attack complex; IRAK, IL-1R-associated kinase.

    Article Snippet: Western blotting was performed with antibodies recognizing the P2X7 receptor (Alomone Lab, Israel; 1:500 dilution), NALP-3 (Alexis Biochem, San Diego, CA; 1:1000 dilution), ASC-1 (Alexis Biochem;1:1000 dilution), caspase-1 (Imgenex, San Diego, CA; 1:1000 dilution), IL-1β (a gift from the National Cancer Institute Biometrics Research Branch (NCIBRB) Preclinical Repository, NCI, Fredrick, MD; 1:10,000 dilution), dysferlin (Novocastra Newcastle on Tyne, UK; 1:500 dilution), and Vinculin (Sigma- Aldrich, St. Louis, MO; 1:2500 dilution) and developed by using Enhanced Chemiluminescence (ECL) (GE Health care, Piscataway, NJ).

    Techniques: Activation Assay, Inhibition

    P2X7 receptor expression is increased in the muscle of SJL/J mice when compared with C57BL/6J control mice. A: Muscle lysates from SJL/J and C57BL/6J mice were examined by Western blotting with P2X7 receptor antibody. The expression of the P2X7 receptor (∼68 kDa) was higher in SJL/J muscle than in C57BL/6J muscle. B: Quantitation of P2X7R expression in muscle lysates from SJL/J ( n = 4) and control C57BL/6J mice ( n = 2). Mean ± SD are shown. * P

    Journal: The American Journal of Pathology

    Article Title: Inflammasome Up-Regulation and Activation in Dysferlin-Deficient Skeletal Muscle

    doi: 10.2353/ajpath.2010.090058

    Figure Lengend Snippet: P2X7 receptor expression is increased in the muscle of SJL/J mice when compared with C57BL/6J control mice. A: Muscle lysates from SJL/J and C57BL/6J mice were examined by Western blotting with P2X7 receptor antibody. The expression of the P2X7 receptor (∼68 kDa) was higher in SJL/J muscle than in C57BL/6J muscle. B: Quantitation of P2X7R expression in muscle lysates from SJL/J ( n = 4) and control C57BL/6J mice ( n = 2). Mean ± SD are shown. * P

    Article Snippet: Western blotting was performed with antibodies recognizing the P2X7 receptor (Alomone Lab, Israel; 1:500 dilution), NALP-3 (Alexis Biochem, San Diego, CA; 1:1000 dilution), ASC-1 (Alexis Biochem;1:1000 dilution), caspase-1 (Imgenex, San Diego, CA; 1:1000 dilution), IL-1β (a gift from the National Cancer Institute Biometrics Research Branch (NCIBRB) Preclinical Repository, NCI, Fredrick, MD; 1:10,000 dilution), dysferlin (Novocastra Newcastle on Tyne, UK; 1:500 dilution), and Vinculin (Sigma- Aldrich, St. Louis, MO; 1:2500 dilution) and developed by using Enhanced Chemiluminescence (ECL) (GE Health care, Piscataway, NJ).

    Techniques: Expressing, Mouse Assay, Western Blot, Quantitation Assay