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  • 95
    Alomone Labs p2x7 receptor intracellular epitope
    Deletion analysis of the mouse P2rx7 gene promoter. A , shown is a schematic diagram of the <t>P2X7</t> promoter constructs consisting of a 5′-flanking region with serial deletions cloned into the pGL4.23 luciferase reporter vector. The arrow shows the direction of transcription. Numbers represent the end points of each construct. B , plasmid constructs were cotransfected with Renilla luciferase vector pGL4.74[hRluc/TK] into N2a cells. 24 h after transfection, cells were harvested, and luciferase activity was measured. Renilla luciferase activity was used to normalize the transfection efficiency. The values represent the mean ± S.E. of at least three independent experiments in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (ANOVA with the post hoc Newman-Keuls test).
    P2x7 Receptor Intracellular Epitope, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 95/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2x7 receptor intracellular epitope/product/Alomone Labs
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    94
    Alomone Labs rabbit anti p2x7 c terminal peptide antibody
    Deletion analysis of the mouse P2rx7 gene promoter. A , shown is a schematic diagram of the <t>P2X7</t> promoter constructs consisting of a 5′-flanking region with serial deletions cloned into the pGL4.23 luciferase reporter vector. The arrow shows the direction of transcription. Numbers represent the end points of each construct. B , plasmid constructs were cotransfected with Renilla luciferase vector pGL4.74[hRluc/TK] into N2a cells. 24 h after transfection, cells were harvested, and luciferase activity was measured. Renilla luciferase activity was used to normalize the transfection efficiency. The values represent the mean ± S.E. of at least three independent experiments in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (ANOVA with the post hoc Newman-Keuls test).
    Rabbit Anti P2x7 C Terminal Peptide Antibody, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/rabbit anti p2x7 c terminal peptide antibody/product/Alomone Labs
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    94
    Alomone Labs mouse receptor
    Deletion analysis of the mouse P2rx7 gene promoter. A , shown is a schematic diagram of the <t>P2X7</t> promoter constructs consisting of a 5′-flanking region with serial deletions cloned into the pGL4.23 luciferase reporter vector. The arrow shows the direction of transcription. Numbers represent the end points of each construct. B , plasmid constructs were cotransfected with Renilla luciferase vector pGL4.74[hRluc/TK] into N2a cells. 24 h after transfection, cells were harvested, and luciferase activity was measured. Renilla luciferase activity was used to normalize the transfection efficiency. The values represent the mean ± S.E. of at least three independent experiments in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (ANOVA with the post hoc Newman-Keuls test).
    Mouse Receptor, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    88
    Alomone Labs p2x 7
    <t>P2X</t> <t>7</t> receptor expression and ATP release of MNL. ( a – c ) show unchallenged MNL examined by FACS and ( d and e ) show leukotoxin-exposed MNL. ( a ) Population identification (R1 and R2) of MNL based on FCS/SSC characteristics. A representative dot-plot is shown. ( b ) Distribution of cells with different surface markers in the two gated populations (R1 and R2). Mean values±S.D. from three different MNL donors are shown. ( c ) Illustration of P2X 7 R expression in R1 and R2 by representative histograms. ( d ) Number of viable cells (PI-negative) in the two different MNL populations (R1 and R2) analyzed by FACS during leukotoxin exposure (10 ng/ml) with and without oATP (500 μ M). ( e ) ATP-release from the macrophages exposed to the leukotoxin (1 and 10 ng/ml)
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    90
    Alomone Labs fluorophore conjugated antibodies p2x7r atto 633
    <t>P2X</t> <t>7</t> receptor expression and ATP release of MNL. ( a – c ) show unchallenged MNL examined by FACS and ( d and e ) show leukotoxin-exposed MNL. ( a ) Population identification (R1 and R2) of MNL based on FCS/SSC characteristics. A representative dot-plot is shown. ( b ) Distribution of cells with different surface markers in the two gated populations (R1 and R2). Mean values±S.D. from three different MNL donors are shown. ( c ) Illustration of P2X 7 R expression in R1 and R2 by representative histograms. ( d ) Number of viable cells (PI-negative) in the two different MNL populations (R1 and R2) analyzed by FACS during leukotoxin exposure (10 ng/ml) with and without oATP (500 μ M). ( e ) ATP-release from the macrophages exposed to the leukotoxin (1 and 10 ng/ml)
    Fluorophore Conjugated Antibodies P2x7r Atto 633, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Image Search Results


    Deletion analysis of the mouse P2rx7 gene promoter. A , shown is a schematic diagram of the P2X7 promoter constructs consisting of a 5′-flanking region with serial deletions cloned into the pGL4.23 luciferase reporter vector. The arrow shows the direction of transcription. Numbers represent the end points of each construct. B , plasmid constructs were cotransfected with Renilla luciferase vector pGL4.74[hRluc/TK] into N2a cells. 24 h after transfection, cells were harvested, and luciferase activity was measured. Renilla luciferase activity was used to normalize the transfection efficiency. The values represent the mean ± S.E. of at least three independent experiments in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (ANOVA with the post hoc Newman-Keuls test).

    Journal: The Journal of Biological Chemistry

    Article Title: The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System

    doi: 10.1074/jbc.M112.390971

    Figure Lengend Snippet: Deletion analysis of the mouse P2rx7 gene promoter. A , shown is a schematic diagram of the P2X7 promoter constructs consisting of a 5′-flanking region with serial deletions cloned into the pGL4.23 luciferase reporter vector. The arrow shows the direction of transcription. Numbers represent the end points of each construct. B , plasmid constructs were cotransfected with Renilla luciferase vector pGL4.74[hRluc/TK] into N2a cells. 24 h after transfection, cells were harvested, and luciferase activity was measured. Renilla luciferase activity was used to normalize the transfection efficiency. The values represent the mean ± S.E. of at least three independent experiments in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (ANOVA with the post hoc Newman-Keuls test).

    Article Snippet: Antibodies used in the study were Sp1 (Merck, catalog #07-645), P2X7 receptor intracellular epitope (Alomone Laboratories, catalog #PR-004), GAPDH (Ambion, catalog #AM4300), NeuN (Chemicon International, catalog #MAB377), GFAP (Santa Cruz Biotechnology, catalog #sc-9065), and Iba1 (Wako, catalog #019-19741).

    Techniques: Construct, Clone Assay, Luciferase, Plasmid Preparation, Transfection, Activity Assay

    Sequence features of the 5′-flanking region of the mouse P2rx7 gene promoter. A , shown is in silico analysis of the putative transcription factor binding sites. A 2334-bp fragment of the 5′-flanking region of the P2rx7 gene was isolated from mouse genomic DNA. Nucleotide numbering is relative to the first nucleotide (adenine +1) of the TSS, which is indicated in a gray background . The sequence lacks TATA and CAAT boxes. The positions of putative transcription factor binding motives identified using the Genomatix MatInspector software tool are boxed. B , shown is a schematic representation of the P2rx7 promoter comprising the region −2114 to +220 bp. CG sites are depicted by black bars . CG content is shown as percentage of the total number of G+C ( top ) and by methylation-susceptible CG pairs, represented by the observed versus expected index ( Obs/Exp ; bottom ).

    Journal: The Journal of Biological Chemistry

    Article Title: The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System

    doi: 10.1074/jbc.M112.390971

    Figure Lengend Snippet: Sequence features of the 5′-flanking region of the mouse P2rx7 gene promoter. A , shown is in silico analysis of the putative transcription factor binding sites. A 2334-bp fragment of the 5′-flanking region of the P2rx7 gene was isolated from mouse genomic DNA. Nucleotide numbering is relative to the first nucleotide (adenine +1) of the TSS, which is indicated in a gray background . The sequence lacks TATA and CAAT boxes. The positions of putative transcription factor binding motives identified using the Genomatix MatInspector software tool are boxed. B , shown is a schematic representation of the P2rx7 promoter comprising the region −2114 to +220 bp. CG sites are depicted by black bars . CG content is shown as percentage of the total number of G+C ( top ) and by methylation-susceptible CG pairs, represented by the observed versus expected index ( Obs/Exp ; bottom ).

    Article Snippet: Antibodies used in the study were Sp1 (Merck, catalog #07-645), P2X7 receptor intracellular epitope (Alomone Laboratories, catalog #PR-004), GAPDH (Ambion, catalog #AM4300), NeuN (Chemicon International, catalog #MAB377), GFAP (Santa Cruz Biotechnology, catalog #sc-9065), and Iba1 (Wako, catalog #019-19741).

    Techniques: Sequencing, In Silico, Binding Assay, Isolation, Software, Methylation

    Functional analyses of putative SP1 elements in the mouse P2rx7 gene promoter. A , shown is a schematic representation of the F fragment (−249 to +220) cloned into the pGL4.23 reporter vector containing the four SP1 binding sites located close to the TSS (+1). F fragment was divided in three subfragments named F1 (−249 to −139), F2 (−148 to −41), and F3 (−50 to +49) as indicated. B , deletion constructs of the whole F fragment were obtained by PCR and cloned into pGL4.23. Numbers represent the end points of each construct. White ellipses represent SP1 sites location. C , plasmid constructs were cotransfected with pGL4.74[hRluc/TK] vector into N2a cells. 24 h after transfection, luciferase activity was measured. Renilla luciferase activity was used to normalize the transfection efficiency. The values represent the mean ± S.E. of at least four independent experiments in triplicate. *, p < 0.05; ***, p < 0.001 versus empty vector; ###, p < 0.001 versus F fragment (ANOVA with the post hoc Newman-Keuls test). D , shown is sequence alignment of the SP1 binding sites located along the mouse P2rx7 gene promoter across different mammalian species. Sequences were obtained from NCBI- GenBank TM and Ensembl databases. Numbers refer to the mouse sequence and ranges from −161 to +22 bp. Alignments of the putative regulatory sites are shown in a gray background. Black bars indicate the percentage of conservation with the mouse sequence. Putative core sequences for the binding of Sp1 are underlined .

    Journal: The Journal of Biological Chemistry

    Article Title: The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System

    doi: 10.1074/jbc.M112.390971

    Figure Lengend Snippet: Functional analyses of putative SP1 elements in the mouse P2rx7 gene promoter. A , shown is a schematic representation of the F fragment (−249 to +220) cloned into the pGL4.23 reporter vector containing the four SP1 binding sites located close to the TSS (+1). F fragment was divided in three subfragments named F1 (−249 to −139), F2 (−148 to −41), and F3 (−50 to +49) as indicated. B , deletion constructs of the whole F fragment were obtained by PCR and cloned into pGL4.23. Numbers represent the end points of each construct. White ellipses represent SP1 sites location. C , plasmid constructs were cotransfected with pGL4.74[hRluc/TK] vector into N2a cells. 24 h after transfection, luciferase activity was measured. Renilla luciferase activity was used to normalize the transfection efficiency. The values represent the mean ± S.E. of at least four independent experiments in triplicate. *, p < 0.05; ***, p < 0.001 versus empty vector; ###, p < 0.001 versus F fragment (ANOVA with the post hoc Newman-Keuls test). D , shown is sequence alignment of the SP1 binding sites located along the mouse P2rx7 gene promoter across different mammalian species. Sequences were obtained from NCBI- GenBank TM and Ensembl databases. Numbers refer to the mouse sequence and ranges from −161 to +22 bp. Alignments of the putative regulatory sites are shown in a gray background. Black bars indicate the percentage of conservation with the mouse sequence. Putative core sequences for the binding of Sp1 are underlined .

    Article Snippet: Antibodies used in the study were Sp1 (Merck, catalog #07-645), P2X7 receptor intracellular epitope (Alomone Laboratories, catalog #PR-004), GAPDH (Ambion, catalog #AM4300), NeuN (Chemicon International, catalog #MAB377), GFAP (Santa Cruz Biotechnology, catalog #sc-9065), and Iba1 (Wako, catalog #019-19741).

    Techniques: Functional Assay, Clone Assay, Plasmid Preparation, Binding Assay, Construct, Transfection, Luciferase, Activity Assay, Sequencing

    SP1c and SP1d binding sites are directly involved in the transcriptional activation of P2rx7 promoter by Sp1 protein. A , shown is a schematic representation of the F fragment (−249 to +220) cloned into the pGL4.23 reporter vector containing four SP1 binding sites located close to the TSS (+1). The position and orientation of SP1 sites are indicated by arrowheads . The sequences of SP1c and SP1d sites are depicted as well as the double point mutations (in gray ) performed to eliminate Sp1-dependent regulation. In N2a cells ( B ) and RAW264.7 cells ( C ) mutation of SP1c and SP1d sites inhibits activation of basal transcription exerted by pP2X7-F2 and pP2X7-F3 constructs, respectively. Reporter activity is shown for pP2X7-F2 ( F2 ), mutated pP2X7-F2 containing the mutation of SP1c site ( F2 mut ), pP2X7-F3 ( F3 ), mutated pP2X7-F3 containing the mutation of SP1d site ( F3 mut ), and empty vector ( pGL4.23 ). D , overexpression of Sp1 protein increases promoter activity induced by pP2X7-F2 construct. The pP2X7-F2 plasmid ( F2 ) containing SP1c site was cotransfected with Sp1 expression plasmid ( Sp1-GFP ) or empty vector (GFP) into N2a cells. Mutation of SP1c binding site ( F2 mut ) reduces promoter activity even in presence when Sp1 is overexpressed. E , shown is overexpression of Sp1 protein increases promoter activity induced by pP2X7-F3 construct. The pP2X7-F3 plasmid ( F3 )-containing SP1d site was cotransfected with Sp1 plasmid ( Sp1-GFP ) or empty vector ( GFP ) into N2a cells. Mutation of SP1d binding site ( F3 mut ) inhibits promoter activity. The values represent the mean ± S.E. of at least three independent experiments in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 (ANOVA with the post hoc Newman-Keuls test).

    Journal: The Journal of Biological Chemistry

    Article Title: The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System

    doi: 10.1074/jbc.M112.390971

    Figure Lengend Snippet: SP1c and SP1d binding sites are directly involved in the transcriptional activation of P2rx7 promoter by Sp1 protein. A , shown is a schematic representation of the F fragment (−249 to +220) cloned into the pGL4.23 reporter vector containing four SP1 binding sites located close to the TSS (+1). The position and orientation of SP1 sites are indicated by arrowheads . The sequences of SP1c and SP1d sites are depicted as well as the double point mutations (in gray ) performed to eliminate Sp1-dependent regulation. In N2a cells ( B ) and RAW264.7 cells ( C ) mutation of SP1c and SP1d sites inhibits activation of basal transcription exerted by pP2X7-F2 and pP2X7-F3 constructs, respectively. Reporter activity is shown for pP2X7-F2 ( F2 ), mutated pP2X7-F2 containing the mutation of SP1c site ( F2 mut ), pP2X7-F3 ( F3 ), mutated pP2X7-F3 containing the mutation of SP1d site ( F3 mut ), and empty vector ( pGL4.23 ). D , overexpression of Sp1 protein increases promoter activity induced by pP2X7-F2 construct. The pP2X7-F2 plasmid ( F2 ) containing SP1c site was cotransfected with Sp1 expression plasmid ( Sp1-GFP ) or empty vector (GFP) into N2a cells. Mutation of SP1c binding site ( F2 mut ) reduces promoter activity even in presence when Sp1 is overexpressed. E , shown is overexpression of Sp1 protein increases promoter activity induced by pP2X7-F3 construct. The pP2X7-F3 plasmid ( F3 )-containing SP1d site was cotransfected with Sp1 plasmid ( Sp1-GFP ) or empty vector ( GFP ) into N2a cells. Mutation of SP1d binding site ( F3 mut ) inhibits promoter activity. The values represent the mean ± S.E. of at least three independent experiments in triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 (ANOVA with the post hoc Newman-Keuls test).

    Article Snippet: Antibodies used in the study were Sp1 (Merck, catalog #07-645), P2X7 receptor intracellular epitope (Alomone Laboratories, catalog #PR-004), GAPDH (Ambion, catalog #AM4300), NeuN (Chemicon International, catalog #MAB377), GFAP (Santa Cruz Biotechnology, catalog #sc-9065), and Iba1 (Wako, catalog #019-19741).

    Techniques: Binding Assay, Activation Assay, Clone Assay, Plasmid Preparation, Mutagenesis, Construct, Activity Assay, Over Expression, Expressing

    Facilitation of endogenous P2X7 receptor expression by Sp1 overexpression. N2a cells were transfected with Sp1 expression plasmid ( Sp1-GFP ) or empty vector ( GFP ) and analyzed 48 h later for Sp1 ( A ) and P2X7 ( B ) mRNA levels by Q-PCR. C , SDS-PAGE was performed to analyze cell lysates from Sp1-transfected N2a cells. Both Sp1 and P2X7 proteins were detected at a size of around 95–105 and 75 kDa, respectively. GAPDH was used as internal loading control. Densitometric analysis was performed using Image J software ( D and E ). The values represent the mean ± S.E. of three independent experiments in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Student's t test).

    Journal: The Journal of Biological Chemistry

    Article Title: The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System

    doi: 10.1074/jbc.M112.390971

    Figure Lengend Snippet: Facilitation of endogenous P2X7 receptor expression by Sp1 overexpression. N2a cells were transfected with Sp1 expression plasmid ( Sp1-GFP ) or empty vector ( GFP ) and analyzed 48 h later for Sp1 ( A ) and P2X7 ( B ) mRNA levels by Q-PCR. C , SDS-PAGE was performed to analyze cell lysates from Sp1-transfected N2a cells. Both Sp1 and P2X7 proteins were detected at a size of around 95–105 and 75 kDa, respectively. GAPDH was used as internal loading control. Densitometric analysis was performed using Image J software ( D and E ). The values represent the mean ± S.E. of three independent experiments in triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Student's t test).

    Article Snippet: Antibodies used in the study were Sp1 (Merck, catalog #07-645), P2X7 receptor intracellular epitope (Alomone Laboratories, catalog #PR-004), GAPDH (Ambion, catalog #AM4300), NeuN (Chemicon International, catalog #MAB377), GFAP (Santa Cruz Biotechnology, catalog #sc-9065), and Iba1 (Wako, catalog #019-19741).

    Techniques: Expressing, Over Expression, Transfection, Plasmid Preparation, SDS Page, Software

    Down-regulation of endogenous P2X7 receptor expression by Sp1 interference. Endogenous Sp1 expression was knocked down in N2a cells using two specific shRNA ( shSP1.1 and shSP1.2 ) and analyzed 48 h later for Sp1 ( A ) and P2X7 ( B ) mRNA level by Q-PCR. Scrambled shRNA was used as negative control. C , silencing of Sp1 reduces both Sp1 and P2X7 proteins expression. SDS-PAGE was performed to analyze cell lysates from N2a cells transfected with shSP1.1, shSP1.2, or scrambled for 72 h. GAPDH was used as internal loading control. Densitometric analysis was performed using Image J software ( D and E ). The values represent the mean ± S.E. of three independent experiments in duplicate or triplicate. **, p < 0.01; ***, p < 0.001 (Student's t test).

    Journal: The Journal of Biological Chemistry

    Article Title: The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System

    doi: 10.1074/jbc.M112.390971

    Figure Lengend Snippet: Down-regulation of endogenous P2X7 receptor expression by Sp1 interference. Endogenous Sp1 expression was knocked down in N2a cells using two specific shRNA ( shSP1.1 and shSP1.2 ) and analyzed 48 h later for Sp1 ( A ) and P2X7 ( B ) mRNA level by Q-PCR. Scrambled shRNA was used as negative control. C , silencing of Sp1 reduces both Sp1 and P2X7 proteins expression. SDS-PAGE was performed to analyze cell lysates from N2a cells transfected with shSP1.1, shSP1.2, or scrambled for 72 h. GAPDH was used as internal loading control. Densitometric analysis was performed using Image J software ( D and E ). The values represent the mean ± S.E. of three independent experiments in duplicate or triplicate. **, p < 0.01; ***, p < 0.001 (Student's t test).

    Article Snippet: Antibodies used in the study were Sp1 (Merck, catalog #07-645), P2X7 receptor intracellular epitope (Alomone Laboratories, catalog #PR-004), GAPDH (Ambion, catalog #AM4300), NeuN (Chemicon International, catalog #MAB377), GFAP (Santa Cruz Biotechnology, catalog #sc-9065), and Iba1 (Wako, catalog #019-19741).

    Techniques: Expressing, shRNA, Negative Control, SDS Page, Transfection, Software

    Sp1-dependent regulation of P2X7 expression in primary cultures of cortical astrocytes and neurons. Primary cultures of cortical astrocytes ( A ) and neurons ( B ) were treated with 300 n m mithramycin A ( MIT ) or vehicle (control) for 24 h. Total RNA was extracted and analyzed for P2X7 mRNA levels by Q-PCR. SDS-PAGE was performed to analyze cell lysates from primary cultures of cortical astrocytes ( C ) and neurons ( D ) treated with 300 n m mithramycin A or vehicle for 48 h. GAPDH was used as internal loading control. E , after 24 h of treatment with mithramycin A or vehicle, total RNA from RAW264.7 cells was extracted and analyzed for P2X7 mRNA expression by Q-PCR. F , cortical astrocytes were transfected with Sp1 expression plasmid ( Sp1-GFP ) or empty vector ( GFP ) and analyzed 48 h later for P2X7 mRNA level by Q-PCR. G , SDS-PAGE was performed to analyze cell lysates from Sp1-transfected astrocytes using anti-P2X7 antibodies. The ratio of P2X7 to GAPDH protein level was calculated by densitometric analysis ( H ). The values represent the mean ± S.E. of three independent experiments in duplicate or triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Student's t test).

    Journal: The Journal of Biological Chemistry

    Article Title: The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System

    doi: 10.1074/jbc.M112.390971

    Figure Lengend Snippet: Sp1-dependent regulation of P2X7 expression in primary cultures of cortical astrocytes and neurons. Primary cultures of cortical astrocytes ( A ) and neurons ( B ) were treated with 300 n m mithramycin A ( MIT ) or vehicle (control) for 24 h. Total RNA was extracted and analyzed for P2X7 mRNA levels by Q-PCR. SDS-PAGE was performed to analyze cell lysates from primary cultures of cortical astrocytes ( C ) and neurons ( D ) treated with 300 n m mithramycin A or vehicle for 48 h. GAPDH was used as internal loading control. E , after 24 h of treatment with mithramycin A or vehicle, total RNA from RAW264.7 cells was extracted and analyzed for P2X7 mRNA expression by Q-PCR. F , cortical astrocytes were transfected with Sp1 expression plasmid ( Sp1-GFP ) or empty vector ( GFP ) and analyzed 48 h later for P2X7 mRNA level by Q-PCR. G , SDS-PAGE was performed to analyze cell lysates from Sp1-transfected astrocytes using anti-P2X7 antibodies. The ratio of P2X7 to GAPDH protein level was calculated by densitometric analysis ( H ). The values represent the mean ± S.E. of three independent experiments in duplicate or triplicate. *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Student's t test).

    Article Snippet: Antibodies used in the study were Sp1 (Merck, catalog #07-645), P2X7 receptor intracellular epitope (Alomone Laboratories, catalog #PR-004), GAPDH (Ambion, catalog #AM4300), NeuN (Chemicon International, catalog #MAB377), GFAP (Santa Cruz Biotechnology, catalog #sc-9065), and Iba1 (Wako, catalog #019-19741).

    Techniques: Expressing, SDS Page, Transfection, Plasmid Preparation

    Expression of P2X7 receptors in macrophages and spleen from adult P2rx7 -EGFP mice. A , representative photomicrographs show the macrophage marker CD11 ( red ) and native EGFP immunofluorescence ( green ) in elicited peritoneal macrophages from 10-week-old P2rx7-EGFP mice. Scale bar = 50 μm. B , photomicrographs show native EGFP immunofluorescence ( green ) in spleen from 10-week-old P2rx7 -EGFP mice. Nuclei are counterstained with DAPI ( blue ). Scale bar = 300 μm. The square in panel B is shown at higher magnification in C. Scale bar = 15 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System

    doi: 10.1074/jbc.M112.390971

    Figure Lengend Snippet: Expression of P2X7 receptors in macrophages and spleen from adult P2rx7 -EGFP mice. A , representative photomicrographs show the macrophage marker CD11 ( red ) and native EGFP immunofluorescence ( green ) in elicited peritoneal macrophages from 10-week-old P2rx7-EGFP mice. Scale bar = 50 μm. B , photomicrographs show native EGFP immunofluorescence ( green ) in spleen from 10-week-old P2rx7 -EGFP mice. Nuclei are counterstained with DAPI ( blue ). Scale bar = 300 μm. The square in panel B is shown at higher magnification in C. Scale bar = 15 μm.

    Article Snippet: Antibodies used in the study were Sp1 (Merck, catalog #07-645), P2X7 receptor intracellular epitope (Alomone Laboratories, catalog #PR-004), GAPDH (Ambion, catalog #AM4300), NeuN (Chemicon International, catalog #MAB377), GFAP (Santa Cruz Biotechnology, catalog #sc-9065), and Iba1 (Wako, catalog #019-19741).

    Techniques: Expressing, Marker, Immunofluorescence

    In vivo colocalization of P2X7 receptor and Sp1 factor in the brain from neonatal P2rx7 -EGFP mice. Representative photomicrographs show native EGFP immunofluorescence ( A and E , green ) and Sp1 immunostaining ( B and F , red ) in different cortical subfields from P2rx7-EGFP mice. Arrows mark the position of EGFP-positive cells. Nuclei are counterstained with DAPI ( C and G , blue ). Merge images are shown ( D and H ). Photomicrographs show native EGFP immunofluorescence ( I , green ) and Sp1 immunostaining ( J , red ) in pons, a brain region with a high P2X7 receptor expression in neonatal P2rx7 -EGFP mice. Arrows indicate EGFP-positive cells. Nuclei are counterstained with DAPI ( K , blue ). A merge image is shown ( L ). Scale bars = 50 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System

    doi: 10.1074/jbc.M112.390971

    Figure Lengend Snippet: In vivo colocalization of P2X7 receptor and Sp1 factor in the brain from neonatal P2rx7 -EGFP mice. Representative photomicrographs show native EGFP immunofluorescence ( A and E , green ) and Sp1 immunostaining ( B and F , red ) in different cortical subfields from P2rx7-EGFP mice. Arrows mark the position of EGFP-positive cells. Nuclei are counterstained with DAPI ( C and G , blue ). Merge images are shown ( D and H ). Photomicrographs show native EGFP immunofluorescence ( I , green ) and Sp1 immunostaining ( J , red ) in pons, a brain region with a high P2X7 receptor expression in neonatal P2rx7 -EGFP mice. Arrows indicate EGFP-positive cells. Nuclei are counterstained with DAPI ( K , blue ). A merge image is shown ( L ). Scale bars = 50 μm.

    Article Snippet: Antibodies used in the study were Sp1 (Merck, catalog #07-645), P2X7 receptor intracellular epitope (Alomone Laboratories, catalog #PR-004), GAPDH (Ambion, catalog #AM4300), NeuN (Chemicon International, catalog #MAB377), GFAP (Santa Cruz Biotechnology, catalog #sc-9065), and Iba1 (Wako, catalog #019-19741).

    Techniques: In Vivo, Immunofluorescence, Immunostaining, Expressing

    Up-regulation of P2X7 receptor expression under serum deprivation. Changes in Sp1 ( A ) and P2X7 ( B ) transcript levels in N2a cells cultured under serum deprivation for 24 or 48 h are shown. In some cases 300 n m mithramycin A ( MIT ) treatment was performed. Total RNA was extracted and analyzed for Sp1 and P2X7 mRNA by Q-PCR. The values represent the mean ± S.E. of three independent experiments in duplicate or triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control (ANOVA with the post hoc Newman-Keuls test); ##, p < 0.01; ###, p < 0.001 (Student's t test). Changes in Sp1 ( C ) and P2X7 ( D ) protein levels under serum deprivation during the whole detection period are shown. SDS-PAGE was performed to analyze Sp1 and P2X7 receptor expression in cell lysates from N2a cells cultured for 0, 24, 48, or 72 h in the absence of serum (FBS). GAPDH was used as internal loading control. The values represent the mean ± S.E. of three independent experiments in duplicate or triplicate. * p < 0.05; ** p < 0.01; *** p < 0.001 versus control (ANOVA with the post hoc Newman-Keuls test). N2a cells were cultured in the presence or absence of serum for 24 or 48 h. Afterward, cells were fixed and immunostained with antibodies against Sp1 ( E and F , red ) or P2X7 ( G and H , red ). Nuclei were labeled with DAPI ( blue ). Scale bar = 50 μm.

    Journal: The Journal of Biological Chemistry

    Article Title: The Specificity Protein Factor Sp1 Mediates Transcriptional Regulation of P2X7 Receptors in the Nervous System

    doi: 10.1074/jbc.M112.390971

    Figure Lengend Snippet: Up-regulation of P2X7 receptor expression under serum deprivation. Changes in Sp1 ( A ) and P2X7 ( B ) transcript levels in N2a cells cultured under serum deprivation for 24 or 48 h are shown. In some cases 300 n m mithramycin A ( MIT ) treatment was performed. Total RNA was extracted and analyzed for Sp1 and P2X7 mRNA by Q-PCR. The values represent the mean ± S.E. of three independent experiments in duplicate or triplicate. * p < 0.05, ** p < 0.01, *** p < 0.001 versus control (ANOVA with the post hoc Newman-Keuls test); ##, p < 0.01; ###, p < 0.001 (Student's t test). Changes in Sp1 ( C ) and P2X7 ( D ) protein levels under serum deprivation during the whole detection period are shown. SDS-PAGE was performed to analyze Sp1 and P2X7 receptor expression in cell lysates from N2a cells cultured for 0, 24, 48, or 72 h in the absence of serum (FBS). GAPDH was used as internal loading control. The values represent the mean ± S.E. of three independent experiments in duplicate or triplicate. * p < 0.05; ** p < 0.01; *** p < 0.001 versus control (ANOVA with the post hoc Newman-Keuls test). N2a cells were cultured in the presence or absence of serum for 24 or 48 h. Afterward, cells were fixed and immunostained with antibodies against Sp1 ( E and F , red ) or P2X7 ( G and H , red ). Nuclei were labeled with DAPI ( blue ). Scale bar = 50 μm.

    Article Snippet: Antibodies used in the study were Sp1 (Merck, catalog #07-645), P2X7 receptor intracellular epitope (Alomone Laboratories, catalog #PR-004), GAPDH (Ambion, catalog #AM4300), NeuN (Chemicon International, catalog #MAB377), GFAP (Santa Cruz Biotechnology, catalog #sc-9065), and Iba1 (Wako, catalog #019-19741).

    Techniques: Expressing, Cell Culture, SDS Page, Labeling

    P2X 7 receptor expression and ATP release of MNL. ( a – c ) show unchallenged MNL examined by FACS and ( d and e ) show leukotoxin-exposed MNL. ( a ) Population identification (R1 and R2) of MNL based on FCS/SSC characteristics. A representative dot-plot is shown. ( b ) Distribution of cells with different surface markers in the two gated populations (R1 and R2). Mean values±S.D. from three different MNL donors are shown. ( c ) Illustration of P2X 7 R expression in R1 and R2 by representative histograms. ( d ) Number of viable cells (PI-negative) in the two different MNL populations (R1 and R2) analyzed by FACS during leukotoxin exposure (10 ng/ml) with and without oATP (500 μ M). ( e ) ATP-release from the macrophages exposed to the leukotoxin (1 and 10 ng/ml)

    Journal: Cell Death & Disease

    Article Title: Cellular and molecular response of human macrophages exposed to Aggregatibacter actinomycetemcomitans leukotoxin

    doi: 10.1038/cddis.2011.6

    Figure Lengend Snippet: P2X 7 receptor expression and ATP release of MNL. ( a – c ) show unchallenged MNL examined by FACS and ( d and e ) show leukotoxin-exposed MNL. ( a ) Population identification (R1 and R2) of MNL based on FCS/SSC characteristics. A representative dot-plot is shown. ( b ) Distribution of cells with different surface markers in the two gated populations (R1 and R2). Mean values±S.D. from three different MNL donors are shown. ( c ) Illustration of P2X 7 R expression in R1 and R2 by representative histograms. ( d ) Number of viable cells (PI-negative) in the two different MNL populations (R1 and R2) analyzed by FACS during leukotoxin exposure (10 ng/ml) with and without oATP (500 μ M). ( e ) ATP-release from the macrophages exposed to the leukotoxin (1 and 10 ng/ml)

    Article Snippet: The two distinct populations (G1 and G2) were characterized with specific fluorescein (FITC)-conjugated antibodies for expression of cell surface receptors: CD14 (Clone TüK4), CD3 (Clone UCHT1) CD19 (Clone HD37) (all from DakoCytomation, Copenhagen, Denmark) and P2X 7 (Rabbit polyclonal) (Alomone Labs, Jerusalem, Israel).

    Techniques: Expressing