p2x7  (Alomone Labs)


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    Structured Review

    Alomone Labs p2x7
    <t>P2X7</t> receptor activity occurs exclusively in HUVEC under atheroprone flow. Representative single cell ATP-induced calcium responses in atheroprotected ( A ) or atheroprone ( B, C ) flow conditioned HUVEC after thapsigargin pre-treatment ± the P2X7 inhibitor 10 µM A438079 (P2X7i) or siRNA control and P2X7 knockdown ( C ) and analysed by measuring the average area under the curve ( n = 4, 175 cells per donor, * indicates P ≤ 0.05 using a paired t -test). Values are mean ± S.E.M.
    P2x7, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2x7/product/Alomone Labs
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    p2x7 - by Bioz Stars, 2022-01
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    Images

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

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

    Journal: Cardiovascular Research

    doi: 10.1093/cvr/cvx213

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

    Techniques Used: Activity Assay

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

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

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

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

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

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

    2) Product Images from "P2X7 Receptor Promotes Mouse Mammary Cancer Cell Invasiveness and Tumour Progression, and Is a Target for Anticancer Treatment"

    Article Title: P2X7 Receptor Promotes Mouse Mammary Cancer Cell Invasiveness and Tumour Progression, and Is a Target for Anticancer Treatment

    Journal: Cancers

    doi: 10.3390/cancers12092342

    P2X7 receptor promotes invadopodial activity of extracellular matrix degradation. ( a ) 4T1 mouse mammary and MDA-MB-435s human cancer cells were grown on Matrigel™ containing DQ-BSA as a fluorogenic substrate for proteases, emitting red fluorescence when degraded. P2X7 receptor was immunodetected using a rabbit primary antibody against P2X7 protein and a secondary ant-rabbit IgG antibody conjugated to AF488. The right panel shows the fluorescence values for the two channels at the locations indicated by a blue arrow. Scale bar, 100 µm. ( b ) Invadopodia entrapped into a 2%-gelatin matrix were fractionated and separated from cytosol and membranes-enriched fractions. The quality of the fractions was assessed by western blotting after SDS-PAGE, using cytosolic (HSC70), membrane (caveolin-1, β-adaptin) and invadopodia (cathepsin B, cortactin, focal adhesion kinase) markers. P2X7 proteins were found to be present in the membrane fractions and enriched in the invadopodia fraction. This figure is representative of 5 independent experiments. The full image blots can be found in Figure S5 . ( c ) The invadopodial activity was assessed as being F-actin foci (green labelling, phalloidin-488) co-localised with focused proteolytic activities (red labelling, DQ-BSA proteolysis) from 4T1 cells grown for 24 h on Matrigel™ containing DQ-BSA in control condition (vehicle), or stimulated with 300 µM BzATP, in the presence and absence of 10 µM A438079. “Coloc” indicates co-localization of degradative activity and F-actin foci and appears as white pixels. Scale bar, 50 µm. ( d ) The number of degradation areas per cell was counted in 20 images, from five independent experiments. * indicates a statistically significant difference at p
    Figure Legend Snippet: P2X7 receptor promotes invadopodial activity of extracellular matrix degradation. ( a ) 4T1 mouse mammary and MDA-MB-435s human cancer cells were grown on Matrigel™ containing DQ-BSA as a fluorogenic substrate for proteases, emitting red fluorescence when degraded. P2X7 receptor was immunodetected using a rabbit primary antibody against P2X7 protein and a secondary ant-rabbit IgG antibody conjugated to AF488. The right panel shows the fluorescence values for the two channels at the locations indicated by a blue arrow. Scale bar, 100 µm. ( b ) Invadopodia entrapped into a 2%-gelatin matrix were fractionated and separated from cytosol and membranes-enriched fractions. The quality of the fractions was assessed by western blotting after SDS-PAGE, using cytosolic (HSC70), membrane (caveolin-1, β-adaptin) and invadopodia (cathepsin B, cortactin, focal adhesion kinase) markers. P2X7 proteins were found to be present in the membrane fractions and enriched in the invadopodia fraction. This figure is representative of 5 independent experiments. The full image blots can be found in Figure S5 . ( c ) The invadopodial activity was assessed as being F-actin foci (green labelling, phalloidin-488) co-localised with focused proteolytic activities (red labelling, DQ-BSA proteolysis) from 4T1 cells grown for 24 h on Matrigel™ containing DQ-BSA in control condition (vehicle), or stimulated with 300 µM BzATP, in the presence and absence of 10 µM A438079. “Coloc” indicates co-localization of degradative activity and F-actin foci and appears as white pixels. Scale bar, 50 µm. ( d ) The number of degradation areas per cell was counted in 20 images, from five independent experiments. * indicates a statistically significant difference at p

    Techniques Used: Activity Assay, Multiple Displacement Amplification, Fluorescence, Western Blot, SDS Page

    P2X7 antagonism slows mammary tumour growth. ( a ) In vivo mouse experiments assessing the effects of treatments with two P2X7 antagonist (A438079 or AZ10606120) on primary tumour growth, in which cancer cells and host organisms both express the P2rx7 gene. ( b ) primary mammary tumour growth represented as a function of the duration of the treatment depending on antagonist treatments (A438079 or AZ10606120) compared to the injection of vehicle. ( c – e ) A Gompertz model was used to assess mammary tumour growth as a function of time for ( c ), vehicle group, ( d ), A438079 and ( e ), AZ10606120. Black, red and blue dots represent individual tumour volumes of these respective categories, blue areas are 90% model prediction intervals and lines are the median tumour growth. Estimated mean (inter-individual standard deviation) of model parameters were k growth = 0.64 day-1 (39%), V max = 1.620 mm 3 (64%), EFF = 0.52 (82%) and γ = 0.17 (–). This analysis showed that tumour volume doubled in a mean time of 0.64 day, that the mean maximum tumour volume was 1.620 mm 3 and that tumour growth was twice slower in the presence of A438079 or AZ10606120 treatment ( p
    Figure Legend Snippet: P2X7 antagonism slows mammary tumour growth. ( a ) In vivo mouse experiments assessing the effects of treatments with two P2X7 antagonist (A438079 or AZ10606120) on primary tumour growth, in which cancer cells and host organisms both express the P2rx7 gene. ( b ) primary mammary tumour growth represented as a function of the duration of the treatment depending on antagonist treatments (A438079 or AZ10606120) compared to the injection of vehicle. ( c – e ) A Gompertz model was used to assess mammary tumour growth as a function of time for ( c ), vehicle group, ( d ), A438079 and ( e ), AZ10606120. Black, red and blue dots represent individual tumour volumes of these respective categories, blue areas are 90% model prediction intervals and lines are the median tumour growth. Estimated mean (inter-individual standard deviation) of model parameters were k growth = 0.64 day-1 (39%), V max = 1.620 mm 3 (64%), EFF = 0.52 (82%) and γ = 0.17 (–). This analysis showed that tumour volume doubled in a mean time of 0.64 day, that the mean maximum tumour volume was 1.620 mm 3 and that tumour growth was twice slower in the presence of A438079 or AZ10606120 treatment ( p

    Techniques Used: In Vivo, Injection, Standard Deviation

    P2X7 receptor in mammary cancer cells enhances primary tumour growth and metastatic development in vivo. ( a ) P2rx7 mRNA expression levels assessed by RT-qPCR in CTL, Crispr#1 and Crispr#2 cell lines, expressed relatively to 4T1 cells. * indicated a statistically significant difference from CTL cells at p
    Figure Legend Snippet: P2X7 receptor in mammary cancer cells enhances primary tumour growth and metastatic development in vivo. ( a ) P2rx7 mRNA expression levels assessed by RT-qPCR in CTL, Crispr#1 and Crispr#2 cell lines, expressed relatively to 4T1 cells. * indicated a statistically significant difference from CTL cells at p

    Techniques Used: In Vivo, Expressing, Quantitative RT-PCR, CRISPR

    P2X7 receptor stimulation promotes the acquisition of a mesenchymal phenotype. ( a ) F-actin cytoskeleton was visualized using phalloidin-488 in 4T1 cells stimulated with 300 µM BzATP at the time indicated. Rapid morphological changes appear as rapidly as after 6 min stimulation. Scale bar, 25 µm. ( b ) The number of new filopodia per cell was counted per hour in cells transfected with the LifeAct plasmid and studied in the presence of 300 µM BzATP, in the presence or absence of 10 µM A438079. * indicates a statistically significant difference at p
    Figure Legend Snippet: P2X7 receptor stimulation promotes the acquisition of a mesenchymal phenotype. ( a ) F-actin cytoskeleton was visualized using phalloidin-488 in 4T1 cells stimulated with 300 µM BzATP at the time indicated. Rapid morphological changes appear as rapidly as after 6 min stimulation. Scale bar, 25 µm. ( b ) The number of new filopodia per cell was counted per hour in cells transfected with the LifeAct plasmid and studied in the presence of 300 µM BzATP, in the presence or absence of 10 µM A438079. * indicates a statistically significant difference at p

    Techniques Used: Transfection, Plasmid Preparation

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    Alomone Labs rabbit anti extracellular p2x7r igg
    The effects of L-NAME and/or PDI knockdown on the amounts of SNO-thiol and total thiol-PDI or <t>-P2X7R,</t> and PDI-P2X7R bindings following SE. As compared to vehicle (V), L-NAME (L) reduces the amount of SNO-thiol-PDI and PDI-P2X7R bindings without affecting PDI expression and the amount of total thiol-PDI under physiological condition. Thus, S -nitrosylation ratio of PDI is decreased. L-NAME abrogates SE-induced alterations in PDI-P2X7R bindings and S -nitrosylation ratio of PDI, except PDI expression. As compared to control siRNA (C), PDI siRNA (P) abolishes the changes in PDI-P2X7R bindings and S -nitrosylation ratio of P2X7R following SE, except its expression. a Representative Western blot for S -nitrosylation and thiolization on PDI and its expression. b Quantification of analyses of S -nitrosylation and thiolization on PDI and its expression. Open circles indicate each individual value. Horizontal bars indicate the mean value. Error bars indicate SEM ( * # p
    Rabbit Anti Extracellular P2x7r Igg, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    The effects of L-NAME and/or PDI knockdown on the amounts of SNO-thiol and total thiol-PDI or -P2X7R, and PDI-P2X7R bindings following SE. As compared to vehicle (V), L-NAME (L) reduces the amount of SNO-thiol-PDI and PDI-P2X7R bindings without affecting PDI expression and the amount of total thiol-PDI under physiological condition. Thus, S -nitrosylation ratio of PDI is decreased. L-NAME abrogates SE-induced alterations in PDI-P2X7R bindings and S -nitrosylation ratio of PDI, except PDI expression. As compared to control siRNA (C), PDI siRNA (P) abolishes the changes in PDI-P2X7R bindings and S -nitrosylation ratio of P2X7R following SE, except its expression. a Representative Western blot for S -nitrosylation and thiolization on PDI and its expression. b Quantification of analyses of S -nitrosylation and thiolization on PDI and its expression. Open circles indicate each individual value. Horizontal bars indicate the mean value. Error bars indicate SEM ( * # p

    Journal: Journal of Neuroinflammation

    Article Title: Protein disulfide isomerase-mediated S-nitrosylation facilitates surface expression of P2X7 receptor following status epilepticus

    doi: 10.1186/s12974-020-02058-y

    Figure Lengend Snippet: The effects of L-NAME and/or PDI knockdown on the amounts of SNO-thiol and total thiol-PDI or -P2X7R, and PDI-P2X7R bindings following SE. As compared to vehicle (V), L-NAME (L) reduces the amount of SNO-thiol-PDI and PDI-P2X7R bindings without affecting PDI expression and the amount of total thiol-PDI under physiological condition. Thus, S -nitrosylation ratio of PDI is decreased. L-NAME abrogates SE-induced alterations in PDI-P2X7R bindings and S -nitrosylation ratio of PDI, except PDI expression. As compared to control siRNA (C), PDI siRNA (P) abolishes the changes in PDI-P2X7R bindings and S -nitrosylation ratio of P2X7R following SE, except its expression. a Representative Western blot for S -nitrosylation and thiolization on PDI and its expression. b Quantification of analyses of S -nitrosylation and thiolization on PDI and its expression. Open circles indicate each individual value. Horizontal bars indicate the mean value. Error bars indicate SEM ( * # p

    Article Snippet: The primary antibodies were mouse anti-PDI (1:1,000, Abcam, ab2792) and rabbit anti-extracellular P2X7R IgG (1:200, Alomone labs, APR-008).

    Techniques: Expressing, Western Blot

    Scheme of the role of PDI-mediated S -nitrosylation of P2X7R in its trafficking to the cell membrane. SE elevates NO synthesis, which S -nitrosylates PDI. SNO-PDI transfers NO to the reduced (immature) P2X7R. In turn, SNO-P2X7R is oxidized by denitrosylases (presumably Trx-1 [ 8 ]), which facilitates its trafficking to the cell surface

    Journal: Journal of Neuroinflammation

    Article Title: Protein disulfide isomerase-mediated S-nitrosylation facilitates surface expression of P2X7 receptor following status epilepticus

    doi: 10.1186/s12974-020-02058-y

    Figure Lengend Snippet: Scheme of the role of PDI-mediated S -nitrosylation of P2X7R in its trafficking to the cell membrane. SE elevates NO synthesis, which S -nitrosylates PDI. SNO-PDI transfers NO to the reduced (immature) P2X7R. In turn, SNO-P2X7R is oxidized by denitrosylases (presumably Trx-1 [ 8 ]), which facilitates its trafficking to the cell surface

    Article Snippet: The primary antibodies were mouse anti-PDI (1:1,000, Abcam, ab2792) and rabbit anti-extracellular P2X7R IgG (1:200, Alomone labs, APR-008).

    Techniques:

    The effects of L-NAME and PDI knockdown on the surface expression of P2X7R. As compared to control animals, SE increases the membrane P2X7R expressions, which are abrogated by both L-NAME and PDI siRNA. a Representative Western blot for surface expression of P2X7R following SE. b Quantification of analyses of P2X7R expression in membrane fractions. Open circles indicate each individual value. Horizontal bars indicate the mean value. Error bars indicate SEM ( * # p

    Journal: Journal of Neuroinflammation

    Article Title: Protein disulfide isomerase-mediated S-nitrosylation facilitates surface expression of P2X7 receptor following status epilepticus

    doi: 10.1186/s12974-020-02058-y

    Figure Lengend Snippet: The effects of L-NAME and PDI knockdown on the surface expression of P2X7R. As compared to control animals, SE increases the membrane P2X7R expressions, which are abrogated by both L-NAME and PDI siRNA. a Representative Western blot for surface expression of P2X7R following SE. b Quantification of analyses of P2X7R expression in membrane fractions. Open circles indicate each individual value. Horizontal bars indicate the mean value. Error bars indicate SEM ( * # p

    Article Snippet: The primary antibodies were mouse anti-PDI (1:1,000, Abcam, ab2792) and rabbit anti-extracellular P2X7R IgG (1:200, Alomone labs, APR-008).

    Techniques: Expressing, Western Blot

    The effects of L-NAME on the amounts of SNO-thiol- and total thiol-P2X7R following SE. As compared to vehicle (V), L-NAME (L) reduces the amount of SNO-thiol-P2X7R and increased that of total thiol-P2X7R without altering P2X7R expression under physiological condition. Thus, S -nitrosylation ratio of P2X7R is decreased. SE upregulates P2X7R expression and S -nitrosylation ratio of P2X7R due to the reduced the amount of total thiol without the altered that of SNO-thiol-P2X7R. L-NAME inhibits these alterations induced by SE, except P2X7R expression. a Representative Western blot for S -nitrosylation and thiolization on P2X7R and its expression. b-e Quantification of analyses of S -nitrosylation and thiolization on P2X7R and its expression. Open circles indicate each individual value. Horizontal bars indicate the mean value. Error bars indicate SEM ( * # p

    Journal: Journal of Neuroinflammation

    Article Title: Protein disulfide isomerase-mediated S-nitrosylation facilitates surface expression of P2X7 receptor following status epilepticus

    doi: 10.1186/s12974-020-02058-y

    Figure Lengend Snippet: The effects of L-NAME on the amounts of SNO-thiol- and total thiol-P2X7R following SE. As compared to vehicle (V), L-NAME (L) reduces the amount of SNO-thiol-P2X7R and increased that of total thiol-P2X7R without altering P2X7R expression under physiological condition. Thus, S -nitrosylation ratio of P2X7R is decreased. SE upregulates P2X7R expression and S -nitrosylation ratio of P2X7R due to the reduced the amount of total thiol without the altered that of SNO-thiol-P2X7R. L-NAME inhibits these alterations induced by SE, except P2X7R expression. a Representative Western blot for S -nitrosylation and thiolization on P2X7R and its expression. b-e Quantification of analyses of S -nitrosylation and thiolization on P2X7R and its expression. Open circles indicate each individual value. Horizontal bars indicate the mean value. Error bars indicate SEM ( * # p

    Article Snippet: The primary antibodies were mouse anti-PDI (1:1,000, Abcam, ab2792) and rabbit anti-extracellular P2X7R IgG (1:200, Alomone labs, APR-008).

    Techniques: Expressing, Western Blot

    Role of FPR2 and P2X7 receptors in senescent and non-senescent endothelial cells. (A) Non-senescent HUVECs were preincubated with WRW4 (0.1 or 1 µ M) or KN-62 (0.1 or 1 µ M) for 30 min, and then incubated with LL-37 (5 µ g/ml) for 24 h. Alternatively, non-senescent HUVECs were preincubated with a combination of WRW4 (0.1 µ M) and KN-62 (0.1 µ M) for 30 min, and then incubated with LL-37 (5 µ g/ml) for 24 h. ICAM-1 protein expression levels were analyzed by western blotting. Relative expression of ICAM-1/GAPDH was calculated as a ratio to LL-37-stimulated cells without antagonists. Data are presented as the mean ± SD of at least three independent experiments. (B) Cell surface expression levels of LL-37 receptors FPR2 and (C) P2X7 were analyzed in senescent and non-senescent HUVECs by flow cytometry. Relative expression in senescent cells was calculated as a ratio to non-senescent cells. Data are presented as the mean ± SD of at least four independent experiments. * P

    Journal: International Journal of Molecular Medicine

    Article Title: Bacterial lipopolysaccharide and antimicrobial LL-37 enhance ICAM-1 expression and NF-κB p65 phosphorylation in senescent endothelial cells

    doi: 10.3892/ijmm.2019.4294

    Figure Lengend Snippet: Role of FPR2 and P2X7 receptors in senescent and non-senescent endothelial cells. (A) Non-senescent HUVECs were preincubated with WRW4 (0.1 or 1 µ M) or KN-62 (0.1 or 1 µ M) for 30 min, and then incubated with LL-37 (5 µ g/ml) for 24 h. Alternatively, non-senescent HUVECs were preincubated with a combination of WRW4 (0.1 µ M) and KN-62 (0.1 µ M) for 30 min, and then incubated with LL-37 (5 µ g/ml) for 24 h. ICAM-1 protein expression levels were analyzed by western blotting. Relative expression of ICAM-1/GAPDH was calculated as a ratio to LL-37-stimulated cells without antagonists. Data are presented as the mean ± SD of at least three independent experiments. (B) Cell surface expression levels of LL-37 receptors FPR2 and (C) P2X7 were analyzed in senescent and non-senescent HUVECs by flow cytometry. Relative expression in senescent cells was calculated as a ratio to non-senescent cells. Data are presented as the mean ± SD of at least four independent experiments. * P

    Article Snippet: Polyclonal anti-P2X7 (cat. no. APR-008; Alomone Labs), normal rabbit IgG (cat. no. PM035; Medical and Biological Laboratories, Ltd.) and PE-conjugated donkey anti-rabbit IgG (cat. no. 406421; BioLegend, Inc.) were also used for flow cytometry.

    Techniques: Incubation, Expressing, Western Blot, Flow Cytometry

    Spinal P2X7Rs are critically involved in the development of morphine tolerance. A – D , Effects of intrathecal injections of A740003 (0.1 nmol), a selective P2X7R antagonist, on the development of morphine tolerance. A , Thermal and ( B ) mechanical nociceptive threshold in CTR- ( n = 7), MS- ( n = 7), MS/A740003- ( n = 7), and A740003- ( n = 6) treated rats. Latency to withdraw from stimulus, tail-flick latency (TFL) and paw withdrawal thresholds (PWTs), are reported as a percentage of the maximum possible effect (MPE). **** p

    Journal: The Journal of Neuroscience

    Article Title: Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance

    doi: 10.1523/JNEUROSCI.0852-17.2017

    Figure Lengend Snippet: Spinal P2X7Rs are critically involved in the development of morphine tolerance. A – D , Effects of intrathecal injections of A740003 (0.1 nmol), a selective P2X7R antagonist, on the development of morphine tolerance. A , Thermal and ( B ) mechanical nociceptive threshold in CTR- ( n = 7), MS- ( n = 7), MS/A740003- ( n = 7), and A740003- ( n = 6) treated rats. Latency to withdraw from stimulus, tail-flick latency (TFL) and paw withdrawal thresholds (PWTs), are reported as a percentage of the maximum possible effect (MPE). **** p

    Article Snippet: Free-floating spinal cord sections were incubated overnight at 4°C in mouse α-CD11b antibody (1:150; CBL1512 EMD, Millipore), rabbit α-P2X7R antibody (1:150; APR-008, Alomone Labs), rabbit α-μ-receptor antibody (1:500; AOR-011, Alomone Labs), rabbit α-Ki67 antibody (1:500; ab16667, Abcam), rabbit α-Iba1 antibody (1:1000; 019-19741, Wako).

    Techniques: Tail Flick Test

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

    Journal: Cardiovascular Research

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

    doi: 10.1093/cvr/cvx213

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

    Article Snippet: 2.1 Antibodies and reagentsSpecific antibodies used, were targeting: P2X7 (APR-008, Alomone); P2X4 (APR-002, Alomone); PDHX (H-130, Santa Cruz); p-p38 Thr180/Tyr182 (28B10, Cell Signalling Technologies); E-selectin (NBP1-45545, Novus Biologicals); CD31-AlexaFluor488 (Clone Mec13.3, Biolegend), and CD39-FITC (Clone A1, Biolegend).

    Techniques: Activity Assay

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

    Journal: Cardiovascular Research

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

    doi: 10.1093/cvr/cvx213

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

    Article Snippet: 2.1 Antibodies and reagentsSpecific antibodies used, were targeting: P2X7 (APR-008, Alomone); P2X4 (APR-002, Alomone); PDHX (H-130, Santa Cruz); p-p38 Thr180/Tyr182 (28B10, Cell Signalling Technologies); E-selectin (NBP1-45545, Novus Biologicals); CD31-AlexaFluor488 (Clone Mec13.3, Biolegend), and CD39-FITC (Clone A1, Biolegend).

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

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

    Journal: Cardiovascular Research

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

    doi: 10.1093/cvr/cvx213

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

    Article Snippet: 2.1 Antibodies and reagentsSpecific antibodies used, were targeting: P2X7 (APR-008, Alomone); P2X4 (APR-002, Alomone); PDHX (H-130, Santa Cruz); p-p38 Thr180/Tyr182 (28B10, Cell Signalling Technologies); E-selectin (NBP1-45545, Novus Biologicals); CD31-AlexaFluor488 (Clone Mec13.3, Biolegend), and CD39-FITC (Clone A1, Biolegend).

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

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

    Journal: Cardiovascular Research

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

    doi: 10.1093/cvr/cvx213

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

    Article Snippet: 2.1 Antibodies and reagentsSpecific antibodies used, were targeting: P2X7 (APR-008, Alomone); P2X4 (APR-002, Alomone); PDHX (H-130, Santa Cruz); p-p38 Thr180/Tyr182 (28B10, Cell Signalling Technologies); E-selectin (NBP1-45545, Novus Biologicals); CD31-AlexaFluor488 (Clone Mec13.3, Biolegend), and CD39-FITC (Clone A1, Biolegend).

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