antibodies against p2x7r  (Alomone Labs)


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    Alomone Labs antibodies against p2x7r
    Immunohistochemical (IHC) staining and IHC-quantitative analysis of <t>P2X7R</t> and P2X4R in the SMGs of CON and DM groups: ( a – d ) IHC images of P2X7R expression in the CON11w, DM11w, CON13w, and DM13w groups. ( e – h ) IHC images of P2X4R in the CON11w, DM11w, CON13w, and DM13w groups. ( i – l ) IHC images of negative control (NC) in the CON11w, DM11w, CON13w, and DM13w groups. Scale bars = 50 μm. ( m ) The P2X7R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. ( n ) The P2X4R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. The data were presented as the means ± SEM (* p < 0.05, *** p < 0.001 and ns not significant).
    Antibodies Against P2x7r, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against p2x7r/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibodies against p2x7r - by Bioz Stars, 2024-06
    86/100 stars

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    1) Product Images from "P2X7R and P2X4R expression of mice submandibular gland in high-fat diet/streptozotocin-induced type 2 diabetes"

    Article Title: P2X7R and P2X4R expression of mice submandibular gland in high-fat diet/streptozotocin-induced type 2 diabetes

    Journal: Scientific Reports

    doi: 10.1038/s41598-024-60519-3

    Immunohistochemical (IHC) staining and IHC-quantitative analysis of P2X7R and P2X4R in the SMGs of CON and DM groups: ( a – d ) IHC images of P2X7R expression in the CON11w, DM11w, CON13w, and DM13w groups. ( e – h ) IHC images of P2X4R in the CON11w, DM11w, CON13w, and DM13w groups. ( i – l ) IHC images of negative control (NC) in the CON11w, DM11w, CON13w, and DM13w groups. Scale bars = 50 μm. ( m ) The P2X7R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. ( n ) The P2X4R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. The data were presented as the means ± SEM (* p < 0.05, *** p < 0.001 and ns not significant).
    Figure Legend Snippet: Immunohistochemical (IHC) staining and IHC-quantitative analysis of P2X7R and P2X4R in the SMGs of CON and DM groups: ( a – d ) IHC images of P2X7R expression in the CON11w, DM11w, CON13w, and DM13w groups. ( e – h ) IHC images of P2X4R in the CON11w, DM11w, CON13w, and DM13w groups. ( i – l ) IHC images of negative control (NC) in the CON11w, DM11w, CON13w, and DM13w groups. Scale bars = 50 μm. ( m ) The P2X7R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. ( n ) The P2X4R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. The data were presented as the means ± SEM (* p < 0.05, *** p < 0.001 and ns not significant).

    Techniques Used: Immunohistochemical staining, Immunohistochemistry, Expressing, Negative Control

    RT-qPCR analysis of P2X7R and P2X4R in the SMG of the CON and DM groups; ( a ) P2X7R mRNA expression in the CON11w, DM11w, CON13w, and DM13w groups (n = 6 each). ( b ) P2X4R mRNA expression in the CON11w, DM11w, CON13w, and DM13w groups (n = 6 each). The data were presented as the SEM (* p < 0.05, ** p < 0.01 and ns not significant).
    Figure Legend Snippet: RT-qPCR analysis of P2X7R and P2X4R in the SMG of the CON and DM groups; ( a ) P2X7R mRNA expression in the CON11w, DM11w, CON13w, and DM13w groups (n = 6 each). ( b ) P2X4R mRNA expression in the CON11w, DM11w, CON13w, and DM13w groups (n = 6 each). The data were presented as the SEM (* p < 0.05, ** p < 0.01 and ns not significant).

    Techniques Used: Quantitative RT-PCR, Expressing

    Primer sequences used in RT-qPCR process.
    Figure Legend Snippet: Primer sequences used in RT-qPCR process.

    Techniques Used: Sequencing

    anti p2x7r  (Alomone Labs)


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    Alomone Labs anti p2x7r
    Anti P2x7r, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p2x7r/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti p2x7r - by Bioz Stars, 2024-06
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    antibodies for p2x7r  (Alomone Labs)


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    Alomone Labs antibodies for p2x7r
    Antibodies For P2x7r, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies for p2x7r/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
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    antibodies for p2x7r - by Bioz Stars, 2024-06
    86/100 stars

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    p2x7r  (Alomone Labs)


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    Alomone Labs p2x7r
    <t> P2X7R </t> SNPs mutations in PDAC patients and controls
    P2x7r, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2x7r/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
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    p2x7r - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer"

    Article Title: Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer

    Journal: Cancer Cell International

    doi: 10.1186/s12935-024-03339-9

     P2X7R  SNPs mutations in PDAC patients and controls
    Figure Legend Snippet: P2X7R SNPs mutations in PDAC patients and controls

    Techniques Used:

    Cell morphology and P2X7R expression in PANC-1 and PSC cells. a Representative fluorescence and transmission images of cells expressing P2X7R + GFP variants. Bars are 50 µm. b Representative WB on whole cell lysate showing P2X7R expression in transfected and non-transfected (NT) cells. GFP and β-actin are also shown as transfection and loading control, respectively
    Figure Legend Snippet: Cell morphology and P2X7R expression in PANC-1 and PSC cells. a Representative fluorescence and transmission images of cells expressing P2X7R + GFP variants. Bars are 50 µm. b Representative WB on whole cell lysate showing P2X7R expression in transfected and non-transfected (NT) cells. GFP and β-actin are also shown as transfection and loading control, respectively

    Techniques Used: Expressing, Fluorescence, Transmission Assay, Transfection

    Effect of P2X7R SNPs on calcium transients in PANC-1, PSCs and HEK293. a Representative recordings of Ca 2+ signals (Fura-2 ratio) in P2X7R + GFP PANC-1 and PSCs (top and bottom panels) transfected with WT (blue), Gly150Arg (orange) and Arg276His (magenta) receptor. The graphs show the response after stimulation with BzATP 100 µM (grey arrow) and the positive control ionomycin 1 µM (green arrow). b Area under the curve (AUC) calculated for 600 s (PANC-1), 400 s (PSCs) and 160 s (HEK293) after the response starts. c Change in Fura-2 (ΔFura-2) between the basal and the maximum peak response to the agonist. Each point represents the change of Fura-2 in a single cell. Comparison between two variants have been evaluated with the non-parametric Mann–Whitney test and the p values are reported in the graphs. Each graph in ( b , c ) shows responses of cells in 3–4 independent experiments and medians and the interquartile range
    Figure Legend Snippet: Effect of P2X7R SNPs on calcium transients in PANC-1, PSCs and HEK293. a Representative recordings of Ca 2+ signals (Fura-2 ratio) in P2X7R + GFP PANC-1 and PSCs (top and bottom panels) transfected with WT (blue), Gly150Arg (orange) and Arg276His (magenta) receptor. The graphs show the response after stimulation with BzATP 100 µM (grey arrow) and the positive control ionomycin 1 µM (green arrow). b Area under the curve (AUC) calculated for 600 s (PANC-1), 400 s (PSCs) and 160 s (HEK293) after the response starts. c Change in Fura-2 (ΔFura-2) between the basal and the maximum peak response to the agonist. Each point represents the change of Fura-2 in a single cell. Comparison between two variants have been evaluated with the non-parametric Mann–Whitney test and the p values are reported in the graphs. Each graph in ( b , c ) shows responses of cells in 3–4 independent experiments and medians and the interquartile range

    Techniques Used: Transfection, Positive Control, Comparison, MANN-WHITNEY

    Reduced ATP-induced dye uptake in PANC-1, PSCs and HEK293 cells expressing Gly150Arg and Arg276His. a Original single-cell traces of the ATP-induced YO-PRO-1 uptake in P2X7R + GFP PANC-1 (top) and PSCs (bottom) expressing WT (blue), Gly150Arg (orange) and Arg276His (magenta) receptors. YO-PRO-1 was added after 25 min (beige arrow) prior to stimulation with 5 mM ATP (green arrow). b Comparison of AUC of P2X7R + GFP expressing the different P2X7R variants. Statistical significance was evaluated with the non-parametric Mann–Whitney test and the p values are reported in the graphs. Graphs in ( b ) and ( c ) include results from single cells obtained in 3–4 independent experiments
    Figure Legend Snippet: Reduced ATP-induced dye uptake in PANC-1, PSCs and HEK293 cells expressing Gly150Arg and Arg276His. a Original single-cell traces of the ATP-induced YO-PRO-1 uptake in P2X7R + GFP PANC-1 (top) and PSCs (bottom) expressing WT (blue), Gly150Arg (orange) and Arg276His (magenta) receptors. YO-PRO-1 was added after 25 min (beige arrow) prior to stimulation with 5 mM ATP (green arrow). b Comparison of AUC of P2X7R + GFP expressing the different P2X7R variants. Statistical significance was evaluated with the non-parametric Mann–Whitney test and the p values are reported in the graphs. Graphs in ( b ) and ( c ) include results from single cells obtained in 3–4 independent experiments

    Techniques Used: Expressing, Comparison, MANN-WHITNEY

    Effects of the P2X7R variants on cell-specific functions: cytokine release from PSCs ( a – d ) and migration of PANC-1 cells ( e ). a – d IL-6, IL-1β, IL-8 and TNF-α released in the supernatant of PSCs expressing WT (blue), Gly150Arg (orange) and Arg276His (magenta) receptors. Stimulation with BzATP 100 µM and inhibition with AZ10606120 (AZ) 10 µM and A438079 (A43) 10 µM are compared to the respective unstimulated controls cells (CTR). The data are shown as the mean ± SEM for IL-6, IL-1β and TNF-α, and median ± interquartile range for IL-8, of n = 9. e Migration of P2X7R + GFP PANC-1 expressing P2X7R variants . Significance has been evaluated as followed: IL-6, IL-1β and TNF-α statistics have been performed with one sample t test, and reported as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; IL-8 statistics have been performed with one-sample Wilcoxon signed rank test and reported as *p < 0.05; **p < 0.01. Migration has been analyzed with the non-parametric Mann–Whitney test and the p values are reported in the graph
    Figure Legend Snippet: Effects of the P2X7R variants on cell-specific functions: cytokine release from PSCs ( a – d ) and migration of PANC-1 cells ( e ). a – d IL-6, IL-1β, IL-8 and TNF-α released in the supernatant of PSCs expressing WT (blue), Gly150Arg (orange) and Arg276His (magenta) receptors. Stimulation with BzATP 100 µM and inhibition with AZ10606120 (AZ) 10 µM and A438079 (A43) 10 µM are compared to the respective unstimulated controls cells (CTR). The data are shown as the mean ± SEM for IL-6, IL-1β and TNF-α, and median ± interquartile range for IL-8, of n = 9. e Migration of P2X7R + GFP PANC-1 expressing P2X7R variants . Significance has been evaluated as followed: IL-6, IL-1β and TNF-α statistics have been performed with one sample t test, and reported as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; IL-8 statistics have been performed with one-sample Wilcoxon signed rank test and reported as *p < 0.05; **p < 0.01. Migration has been analyzed with the non-parametric Mann–Whitney test and the p values are reported in the graph

    Techniques Used: Migration, Expressing, Inhibition, MANN-WHITNEY

    p2x7r  (Alomone Labs)


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    Alomone Labs p2x7r
    P2x7r, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2x7r/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
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    p2x7r - by Bioz Stars, 2024-06
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    antibodies against p2x7r  (Alomone Labs)


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    Alomone Labs antibodies against p2x7r
    <t>P2X7R</t> expression was elevated in the myocardium of Ang II-infused mice and in cardiomyocytes. A Heatmap of P2X7R expression profiles identified in mouse models following Ang II infusion from the GSE89712 dataset (n = 2). B Representative immunoblot showing P2X7R in the myocardium of Ang II-infused mice. C mRNA levels of P2X7R in cardiac tissues. D Double immunofluorescence staining for P2X7R (red), the fibrosis marker vimentin (green, the top two rows) or the myocyte marker α-actin (green, the next two rows) in the myocardium of Ang II-infused mice. Merged images (orange) showing colocalization. E Quantification of the P2X7R immunoreactive area (%) in D . F Quantification of double immunoreactivity showing the percentages of P2X7R-positive plus α-actin- and vimentin-positive areas in images of Ang II-infused mice in D . G Representative immunoblot showing P2X7R in primary cardiomyocytes, H9c2 cardiomyocyte-like cell lines and primary fibroblasts stimulated with different concentrations of Ang II (n = 6; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Vimentin group). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Antibodies Against P2x7r, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/antibodies against p2x7r/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    antibodies against p2x7r - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R"

    Article Title: Angiotension II directly bind P2X7 receptor to induce myocardial ferroptosis and remodeling by activating human antigen R

    Journal: Redox Biology

    doi: 10.1016/j.redox.2024.103154

    P2X7R expression was elevated in the myocardium of Ang II-infused mice and in cardiomyocytes. A Heatmap of P2X7R expression profiles identified in mouse models following Ang II infusion from the GSE89712 dataset (n = 2). B Representative immunoblot showing P2X7R in the myocardium of Ang II-infused mice. C mRNA levels of P2X7R in cardiac tissues. D Double immunofluorescence staining for P2X7R (red), the fibrosis marker vimentin (green, the top two rows) or the myocyte marker α-actin (green, the next two rows) in the myocardium of Ang II-infused mice. Merged images (orange) showing colocalization. E Quantification of the P2X7R immunoreactive area (%) in D . F Quantification of double immunoreactivity showing the percentages of P2X7R-positive plus α-actin- and vimentin-positive areas in images of Ang II-infused mice in D . G Representative immunoblot showing P2X7R in primary cardiomyocytes, H9c2 cardiomyocyte-like cell lines and primary fibroblasts stimulated with different concentrations of Ang II (n = 6; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Vimentin group). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: P2X7R expression was elevated in the myocardium of Ang II-infused mice and in cardiomyocytes. A Heatmap of P2X7R expression profiles identified in mouse models following Ang II infusion from the GSE89712 dataset (n = 2). B Representative immunoblot showing P2X7R in the myocardium of Ang II-infused mice. C mRNA levels of P2X7R in cardiac tissues. D Double immunofluorescence staining for P2X7R (red), the fibrosis marker vimentin (green, the top two rows) or the myocyte marker α-actin (green, the next two rows) in the myocardium of Ang II-infused mice. Merged images (orange) showing colocalization. E Quantification of the P2X7R immunoreactive area (%) in D . F Quantification of double immunoreactivity showing the percentages of P2X7R-positive plus α-actin- and vimentin-positive areas in images of Ang II-infused mice in D . G Representative immunoblot showing P2X7R in primary cardiomyocytes, H9c2 cardiomyocyte-like cell lines and primary fibroblasts stimulated with different concentrations of Ang II (n = 6; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Vimentin group). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Expressing, Western Blot, Double Immunofluorescence Staining, Marker

    P2X7R deficiency improved Ang II-induced cardiac diastolic and systolic dysfunction and cardiac remodeling in mice. A C57BL/6 or P2X7R knockout mice were infused with Ang II or saline for 4 weeks. Heart weight-to-body weight ratio (HW/BW) in each group of mice. B Representative M-mode echocardiographic images. C–F Echocardiographic analysis of the ejection fraction (EF, C ), fractional shortening (FS, D ), left ventricular posterior wall thickness in diastole (LVPWd, E ) and interventricular septal thickness at diastole (IVSd, F ). G The serum level of ANP was detected with an ELISA kit. H Representative H&E-stained images (longitudinal section, upper and transverse section, bottom). I and J Representative images of WGA in transverse sections of heart tissues ( I ) and quantification of the cardiomyocyte area from WGA ( J ). K and L Representative images of Masson staining ( K ) and quantification of the interstitial fibrotic area from Masson staining ( L ). M Representative Western blot analysis of β-MYHC, ANP, COL-1, MMP9 and TGF-β in cardiac tissues; GAPDH was used as a loading control. N Densitometric quantification of immunoblots in M (n = 6; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II group).
    Figure Legend Snippet: P2X7R deficiency improved Ang II-induced cardiac diastolic and systolic dysfunction and cardiac remodeling in mice. A C57BL/6 or P2X7R knockout mice were infused with Ang II or saline for 4 weeks. Heart weight-to-body weight ratio (HW/BW) in each group of mice. B Representative M-mode echocardiographic images. C–F Echocardiographic analysis of the ejection fraction (EF, C ), fractional shortening (FS, D ), left ventricular posterior wall thickness in diastole (LVPWd, E ) and interventricular septal thickness at diastole (IVSd, F ). G The serum level of ANP was detected with an ELISA kit. H Representative H&E-stained images (longitudinal section, upper and transverse section, bottom). I and J Representative images of WGA in transverse sections of heart tissues ( I ) and quantification of the cardiomyocyte area from WGA ( J ). K and L Representative images of Masson staining ( K ) and quantification of the interstitial fibrotic area from Masson staining ( L ). M Representative Western blot analysis of β-MYHC, ANP, COL-1, MMP9 and TGF-β in cardiac tissues; GAPDH was used as a loading control. N Densitometric quantification of immunoblots in M (n = 6; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II group).

    Techniques Used: Knock-Out, Saline, Enzyme-linked Immunosorbent Assay, Staining, Western Blot

    P2X7R deficiency alleviates Ang II-induced myocardial ferroptosis in Ang II-induced mice. A Representative Western blot analysis of HO-1 and GPX4 levels in the heart tissues of mice. GAPDH was used as a loading control. B Densitometric quantification of immunoblots in A. C and D mRNA levels of HO-1 and GPX4 in heart tissues. E The contents of Fe 2+ in heart tissues measured by a kit. F Representative immunohistochemical images of 4-HNE in heart tissues. G Ultrastructural changes, including decreased mitochondrial volume, increased bilayer membrane density and the disappearance of mitochondrial cristae, were detected by transmission electron microscopy (TEM). H and I C57BL/6 or P2X7R knockout mice were fed a high-iron diet and infused with Ang II or saline for 4 weeks. Echocardiographic analysis of ejection fraction (EF, H ), fractional shortening (FS, I ) J-L Representative images of H&E ( J ) and Masson staining ( K ); quantification of interstitial fibrotic area from Masson staining ( L ) (n = 6; *P < 0.05, **P < 0.01 versus Ctrl group; #P < 0.05 versus Ang II group).
    Figure Legend Snippet: P2X7R deficiency alleviates Ang II-induced myocardial ferroptosis in Ang II-induced mice. A Representative Western blot analysis of HO-1 and GPX4 levels in the heart tissues of mice. GAPDH was used as a loading control. B Densitometric quantification of immunoblots in A. C and D mRNA levels of HO-1 and GPX4 in heart tissues. E The contents of Fe 2+ in heart tissues measured by a kit. F Representative immunohistochemical images of 4-HNE in heart tissues. G Ultrastructural changes, including decreased mitochondrial volume, increased bilayer membrane density and the disappearance of mitochondrial cristae, were detected by transmission electron microscopy (TEM). H and I C57BL/6 or P2X7R knockout mice were fed a high-iron diet and infused with Ang II or saline for 4 weeks. Echocardiographic analysis of ejection fraction (EF, H ), fractional shortening (FS, I ) J-L Representative images of H&E ( J ) and Masson staining ( K ); quantification of interstitial fibrotic area from Masson staining ( L ) (n = 6; *P < 0.05, **P < 0.01 versus Ctrl group; #P < 0.05 versus Ang II group).

    Techniques Used: Western Blot, Immunohistochemical staining, Membrane, Transmission Assay, Electron Microscopy, Knock-Out, Saline, Staining

    P2X7R inhibition abolished Ang II-induced ferroptosis in vitro. H9c2 cells were pretreated with 10 μM A438079 (a P2X7R inhibitor) for 2 h and then stimulated with 1 μM Ang II for 24 h. A Representative Western blot analysis of HO-1 and GPX4 levels; GAPDH was used as a loading control. B and C Densitometric quantification of immunoblots in A . D-F Representative MDA content, SOD activity and GSH levels in each group. G The mitochondrial membrane potential of H9c2 cells was measured by JC-1 staining (n = 3; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II group).
    Figure Legend Snippet: P2X7R inhibition abolished Ang II-induced ferroptosis in vitro. H9c2 cells were pretreated with 10 μM A438079 (a P2X7R inhibitor) for 2 h and then stimulated with 1 μM Ang II for 24 h. A Representative Western blot analysis of HO-1 and GPX4 levels; GAPDH was used as a loading control. B and C Densitometric quantification of immunoblots in A . D-F Representative MDA content, SOD activity and GSH levels in each group. G The mitochondrial membrane potential of H9c2 cells was measured by JC-1 staining (n = 3; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II group).

    Techniques Used: Inhibition, In Vitro, Western Blot, Activity Assay, Membrane, Staining

    P2X7R blockade affects the stability of GPX4 and HO-1 mRNA by regulating HuR expression and nucleocytoplasmic shuttling. A and B H9c2 cells transfected with si-P2X7R were stimulated with 1 μM Ang Ⅱ for 24 h, and actinomycin D (Act D) was added at different times to interfere with the cell transcriptional cycle. The mRNA levels of GPX4 ( A ) and HO-1 ( B ) in H9c2 cells were detected. C Representative Western blot analysis of HuR levels with GAPDH as a loading control. D Densitometric quantification of immunoblots in C . E Immunofluorescence staining for HuR (green) in H9c2 cells. Increased fluorescence intensity in the cytoplasm after stimulation with 1 μM Ang Ⅱ. F and G, Relative GPX4 mRNA ( F ) and HO-1 mRNA ( G ) in H9c2 cells transfected with Si-HuR, stimulated with Ang Ⅱ and then treated with Act D. H and I RNA binding protein immunoprecipitation assays were used to verify the binding of HuR to GPX4 ( H ) and HO-1 ( I ) mRNA (n = 3; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II group). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: P2X7R blockade affects the stability of GPX4 and HO-1 mRNA by regulating HuR expression and nucleocytoplasmic shuttling. A and B H9c2 cells transfected with si-P2X7R were stimulated with 1 μM Ang Ⅱ for 24 h, and actinomycin D (Act D) was added at different times to interfere with the cell transcriptional cycle. The mRNA levels of GPX4 ( A ) and HO-1 ( B ) in H9c2 cells were detected. C Representative Western blot analysis of HuR levels with GAPDH as a loading control. D Densitometric quantification of immunoblots in C . E Immunofluorescence staining for HuR (green) in H9c2 cells. Increased fluorescence intensity in the cytoplasm after stimulation with 1 μM Ang Ⅱ. F and G, Relative GPX4 mRNA ( F ) and HO-1 mRNA ( G ) in H9c2 cells transfected with Si-HuR, stimulated with Ang Ⅱ and then treated with Act D. H and I RNA binding protein immunoprecipitation assays were used to verify the binding of HuR to GPX4 ( H ) and HO-1 ( I ) mRNA (n = 3; *P < 0.05, **P < 0.01 versus the Ctrl group; #P < 0.05 versus the Ang II group). (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Expressing, Transfection, Western Blot, Immunofluorescence, Staining, Fluorescence, RNA Binding Assay, Immunoprecipitation, Binding Assay

    Ang II directly binds to the P2X7R protein at positions LYS-66 and MET-212. A and B H9c2 cells were treated with 1 μM Ang II or biotinylated angiotensin II (Bio-Ang II) for 24 h. Representative Western blot analysis of P2X7R in cells with GAPDH as a loading control ( A ). Densitometric quantification of immunoblots in A ( B ) (n = 3; **P < 0.01 versus the Ctrl group). C H9c2 cells were treated with 1 μM Bio-Ang II or free biotin for 24 h, followed by double-immunofluorescence staining for biotin (green) and P2X7R (red). D and F Binding of Bio-Ang II to P2X7R was determined by pull-down assays. Bio-Ang II was added to streptavidin-agarose beads, and total lysates were used as an input control. Lysates prepared from heart tissues of mice infused with saline or Ang II were added to streptavidin-agarose beads with Bio-Ang II ( D ). Lysates were prepared from primary cardiomyocytes overexpressing Flag-P2X7R. Untreated beads (Blank), biotin alone (Bio) and unconjugated Ang II (Ang II) were used as controls ( E ). F Biolayer interferometry (BLI) analysis of the binding of Ang II to purified P2X7R protein. Ang II was added at different concentrations, and kinetic analysis was performed (shown in the panel below). G and H Molecular docking study between compound Ang II and the 3D structure of P2RX7 (PDB code: 6U9W ) ( G ). The key amino acids are connected to the molecular formula of Ang II through dashed lines ( H ). I HEK-293T cells were transfected with Flag-tagged P2X7R with THR-189 (Flag-PT189), Flag-tagged P2X7R with LYS-66 (Flag-PL66), Flag-tagged P2X7R with LYS-193 (Flag-PL193) and Flag-tagged P2RX7 with MET-212 (Flag-PM212). Lysates were added to streptavidin-agarose beads with Bio-Ang II. H Schematic representation of the key findings of this study. Ang II-induced myocardial remodeling involves upregulation of P2X7R expression and activation of ferroptosis. Intriguingly, Ang II directly interacts with the P2X7R protein. Furthermore, P2X7R mediates Ang II-induced myocardial ferroptosis through HuR, which affects the stability of GPX4 and HO-1 mRNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)
    Figure Legend Snippet: Ang II directly binds to the P2X7R protein at positions LYS-66 and MET-212. A and B H9c2 cells were treated with 1 μM Ang II or biotinylated angiotensin II (Bio-Ang II) for 24 h. Representative Western blot analysis of P2X7R in cells with GAPDH as a loading control ( A ). Densitometric quantification of immunoblots in A ( B ) (n = 3; **P < 0.01 versus the Ctrl group). C H9c2 cells were treated with 1 μM Bio-Ang II or free biotin for 24 h, followed by double-immunofluorescence staining for biotin (green) and P2X7R (red). D and F Binding of Bio-Ang II to P2X7R was determined by pull-down assays. Bio-Ang II was added to streptavidin-agarose beads, and total lysates were used as an input control. Lysates prepared from heart tissues of mice infused with saline or Ang II were added to streptavidin-agarose beads with Bio-Ang II ( D ). Lysates were prepared from primary cardiomyocytes overexpressing Flag-P2X7R. Untreated beads (Blank), biotin alone (Bio) and unconjugated Ang II (Ang II) were used as controls ( E ). F Biolayer interferometry (BLI) analysis of the binding of Ang II to purified P2X7R protein. Ang II was added at different concentrations, and kinetic analysis was performed (shown in the panel below). G and H Molecular docking study between compound Ang II and the 3D structure of P2RX7 (PDB code: 6U9W ) ( G ). The key amino acids are connected to the molecular formula of Ang II through dashed lines ( H ). I HEK-293T cells were transfected with Flag-tagged P2X7R with THR-189 (Flag-PT189), Flag-tagged P2X7R with LYS-66 (Flag-PL66), Flag-tagged P2X7R with LYS-193 (Flag-PL193) and Flag-tagged P2RX7 with MET-212 (Flag-PM212). Lysates were added to streptavidin-agarose beads with Bio-Ang II. H Schematic representation of the key findings of this study. Ang II-induced myocardial remodeling involves upregulation of P2X7R expression and activation of ferroptosis. Intriguingly, Ang II directly interacts with the P2X7R protein. Furthermore, P2X7R mediates Ang II-induced myocardial ferroptosis through HuR, which affects the stability of GPX4 and HO-1 mRNA. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

    Techniques Used: Western Blot, Double Immunofluorescence Staining, Binding Assay, Saline, Purification, Transfection, Expressing, Activation Assay

    antibodies against p2x7r  (Alomone Labs)


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    Alomone Labs antibodies against p2x7r
    Antibodies Against P2x7r, 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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    anti p2x7r c terminal  (Alomone Labs)


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    Alomone Labs anti p2x7r c terminal
    Overexpression of NFATc1 upregulates <t>P2X7R</t> expression in human IVD cells. IVD cells were transfected with 100 ng or 200 ng of pRSV-NFATc1/A expression vector (NFATc1) for 16 h and treated with PMA (20 nM) and ionomycin (1 μM) for 3 h. Cells were collected and subjected to RNA and protein analysis. mRNA levels of NFATc1 and P2X7R were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.01; ** p < 0.0001. Protein levels of NFATc1 and P2X7R were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.05. Scale bar = 20 μm.
    Anti P2x7r C Terminal, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p2x7r c terminal/product/Alomone Labs
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    1) Product Images from "The NFATc1/P2X7 receptor relationship in human intervertebral disc cells"

    Article Title: The NFATc1/P2X7 receptor relationship in human intervertebral disc cells

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2024.1368318

    Overexpression of NFATc1 upregulates P2X7R expression in human IVD cells. IVD cells were transfected with 100 ng or 200 ng of pRSV-NFATc1/A expression vector (NFATc1) for 16 h and treated with PMA (20 nM) and ionomycin (1 μM) for 3 h. Cells were collected and subjected to RNA and protein analysis. mRNA levels of NFATc1 and P2X7R were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.01; ** p < 0.0001. Protein levels of NFATc1 and P2X7R were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.05. Scale bar = 20 μm.
    Figure Legend Snippet: Overexpression of NFATc1 upregulates P2X7R expression in human IVD cells. IVD cells were transfected with 100 ng or 200 ng of pRSV-NFATc1/A expression vector (NFATc1) for 16 h and treated with PMA (20 nM) and ionomycin (1 μM) for 3 h. Cells were collected and subjected to RNA and protein analysis. mRNA levels of NFATc1 and P2X7R were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.01; ** p < 0.0001. Protein levels of NFATc1 and P2X7R were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.05. Scale bar = 20 μm.

    Techniques Used: Over Expression, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunocytochemistry, Immunostaining, Software

    Association between P2X7R and NFATc1. (A) Immunofluorescence detection of P2X7R and NFATc1. The IVD cells were co-labeled with anti-P2X7R C-terminal antibody (green) and anti-NFATc1 antibody (red). Nuclei were counterstained with DAPI (blue). Representative images together with high-magnification pictures are reported (the nucleus chosen for magnification is indicated with a white arrowhead). Merge images represent an overlay of the two channels where colocalization is indicated by a color change (yellow). The graph indicates the fluorescence intensity profile along the white arrow. The blue, red, and green lines correspond to signals of DNA, NFATc1, and P2X7R, respectively. Scale bar = 10 μm; high-magnification pictures, scale bar = 2 μm. (B) CoIP assay to evaluate the interaction between NFATc1 and P2X7R. Immunoprecipitations of NFATc1 followed by immunoblot analyses with anti-NFATc1 and anti-P2X7R antibodies were performed in IVD cells derived from four patients with the same grade of degeneration pooled together. The negative control of immunoprecipitation (normal mouse IgG) was also included. (C) PLA of P2X7R and NFATc1 interactions performed in IVD cells using primary antibody against P2X7R and NFATc1. Detection of P2X7R was performed by immunofluorescence (green). Interactions between P2X7R and NFATc1 are revealed as red dots. Nuclei were counterstained with DAPI (blue). Quantification of positive PLA signals per cell is reported as a dot plot with mean (±SD, n = 3). PLA dot counts per cytoplasm and nucleus are reported separately. Data analyzed were based on an average of 100 cells. Scale bar = 10 μm; high-magnification pictures, scale bar = 5 μm. (D) Immunofluorescence detection of P2X7R and lamin A/C. The IVD cells were co-labeled with anti-P2X7R C-terminal antibody (green) and anti-lamin A/C antibody (red). Nuclei were counterstained with DAPI (blue). Representative images together with high-magnification pictures are reported (the nucleus chosen for magnification is indicated with a white arrowhead). Merge images represent an overlay of the two channels where colocalization is indicated by a color change (yellow). The graph indicates the fluorescence intensity profile along the white arrow. The blue, red, and green lines correspond to signals of DNA, lamin A/C, and P2X7R, respectively. Scale bar = 10 μm; high-magnification pictures, scale bar = 2 μm. (E) PLA of P2X7R and lamin A/C interactions performed in IVD cells using a primary antibody against P2X7R and lamin A/C. Detection of lamin A/C was performed by immunofluorescence (green). Interactions between P2X7R and lamin A/C are revealed as red dots. Nuclei were counterstained with DAPI (blue). Quantification of positive PLA signals per nucleus is reported as dot plot with mean (±SD, n = 3). Data analyzed were based on an average of 100 cells. Scale bar = 10 μm; high-magnification pictures, scale bar = 3 μm.
    Figure Legend Snippet: Association between P2X7R and NFATc1. (A) Immunofluorescence detection of P2X7R and NFATc1. The IVD cells were co-labeled with anti-P2X7R C-terminal antibody (green) and anti-NFATc1 antibody (red). Nuclei were counterstained with DAPI (blue). Representative images together with high-magnification pictures are reported (the nucleus chosen for magnification is indicated with a white arrowhead). Merge images represent an overlay of the two channels where colocalization is indicated by a color change (yellow). The graph indicates the fluorescence intensity profile along the white arrow. The blue, red, and green lines correspond to signals of DNA, NFATc1, and P2X7R, respectively. Scale bar = 10 μm; high-magnification pictures, scale bar = 2 μm. (B) CoIP assay to evaluate the interaction between NFATc1 and P2X7R. Immunoprecipitations of NFATc1 followed by immunoblot analyses with anti-NFATc1 and anti-P2X7R antibodies were performed in IVD cells derived from four patients with the same grade of degeneration pooled together. The negative control of immunoprecipitation (normal mouse IgG) was also included. (C) PLA of P2X7R and NFATc1 interactions performed in IVD cells using primary antibody against P2X7R and NFATc1. Detection of P2X7R was performed by immunofluorescence (green). Interactions between P2X7R and NFATc1 are revealed as red dots. Nuclei were counterstained with DAPI (blue). Quantification of positive PLA signals per cell is reported as a dot plot with mean (±SD, n = 3). PLA dot counts per cytoplasm and nucleus are reported separately. Data analyzed were based on an average of 100 cells. Scale bar = 10 μm; high-magnification pictures, scale bar = 5 μm. (D) Immunofluorescence detection of P2X7R and lamin A/C. The IVD cells were co-labeled with anti-P2X7R C-terminal antibody (green) and anti-lamin A/C antibody (red). Nuclei were counterstained with DAPI (blue). Representative images together with high-magnification pictures are reported (the nucleus chosen for magnification is indicated with a white arrowhead). Merge images represent an overlay of the two channels where colocalization is indicated by a color change (yellow). The graph indicates the fluorescence intensity profile along the white arrow. The blue, red, and green lines correspond to signals of DNA, lamin A/C, and P2X7R, respectively. Scale bar = 10 μm; high-magnification pictures, scale bar = 2 μm. (E) PLA of P2X7R and lamin A/C interactions performed in IVD cells using a primary antibody against P2X7R and lamin A/C. Detection of lamin A/C was performed by immunofluorescence (green). Interactions between P2X7R and lamin A/C are revealed as red dots. Nuclei were counterstained with DAPI (blue). Quantification of positive PLA signals per nucleus is reported as dot plot with mean (±SD, n = 3). Data analyzed were based on an average of 100 cells. Scale bar = 10 μm; high-magnification pictures, scale bar = 3 μm.

    Techniques Used: Immunofluorescence, Labeling, Fluorescence, Co-Immunoprecipitation Assay, Western Blot, Derivative Assay, Negative Control, Immunoprecipitation

    Regulation of NFATc1 and P2X7R expression under hypoxia. (A) HIF-1α expression was analyzed by immunocytochemistry in IVD cells during the de-differentiation process from P0 to P2 and in IVD cells cultured in the absence (untreated) or presence of CoCl 2 for 24 h. Representative photomicrographs are reported. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to P0 cells or untreated cells. Data are presented as the mean ± SD (n = 4). * p < 0.0001 P2 vs P0; ** p < 0.05 CoCl 2 vs untreated. Scale bar = 20 μm. (B, C) IVD cells were cultured in the absence (untreated) or presence of CoCl 2 for 24 h. Cells were then collected and subjected to RNA (B) and protein (C) analyses. mRNA levels of P2X7R and NFATc1 were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.0001 CoCl 2 vs untreated. Protein levels were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.0001. Scale bar = 20 μm. (D) ChIP analysis was performed in IVD cells cultured in the absence (untreated) or presence of CoCl 2 . The recognition motifs of HIF-1α from the JASPAR database and the positioning of potential hypoxia-responsive elements (HREs) within the P2RX7 (−2000/+32) promoter region are reported (HREs, triangle; NFATc1 potential binding sites, gray rectangles). The positions of specific primers used for qPCR amplification are also reported. Results of qPCR were analyzed using the 2 −ΔΔCt method, normalized for the input signal, and presented as fold enrichment with respect to the IgG negative control. Data are expressed as the mean ± SD (n = 2).
    Figure Legend Snippet: Regulation of NFATc1 and P2X7R expression under hypoxia. (A) HIF-1α expression was analyzed by immunocytochemistry in IVD cells during the de-differentiation process from P0 to P2 and in IVD cells cultured in the absence (untreated) or presence of CoCl 2 for 24 h. Representative photomicrographs are reported. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to P0 cells or untreated cells. Data are presented as the mean ± SD (n = 4). * p < 0.0001 P2 vs P0; ** p < 0.05 CoCl 2 vs untreated. Scale bar = 20 μm. (B, C) IVD cells were cultured in the absence (untreated) or presence of CoCl 2 for 24 h. Cells were then collected and subjected to RNA (B) and protein (C) analyses. mRNA levels of P2X7R and NFATc1 were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.0001 CoCl 2 vs untreated. Protein levels were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.0001. Scale bar = 20 μm. (D) ChIP analysis was performed in IVD cells cultured in the absence (untreated) or presence of CoCl 2 . The recognition motifs of HIF-1α from the JASPAR database and the positioning of potential hypoxia-responsive elements (HREs) within the P2RX7 (−2000/+32) promoter region are reported (HREs, triangle; NFATc1 potential binding sites, gray rectangles). The positions of specific primers used for qPCR amplification are also reported. Results of qPCR were analyzed using the 2 −ΔΔCt method, normalized for the input signal, and presented as fold enrichment with respect to the IgG negative control. Data are expressed as the mean ± SD (n = 2).

    Techniques Used: Expressing, Immunocytochemistry, Cell Culture, Software, Quantitative RT-PCR, Immunostaining, Binding Assay, Amplification, Negative Control

    anti p2x7r c terminal  (Alomone Labs)


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    Alomone Labs anti p2x7r c terminal
    Overexpression of NFATc1 upregulates <t>P2X7R</t> expression in human IVD cells. IVD cells were transfected with 100 ng or 200 ng of pRSV-NFATc1/A expression vector (NFATc1) for 16 h and treated with PMA (20 nM) and ionomycin (1 μM) for 3 h. Cells were collected and subjected to RNA and protein analysis. mRNA levels of NFATc1 and P2X7R were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.01; ** p < 0.0001. Protein levels of NFATc1 and P2X7R were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.05. Scale bar = 20 μm.
    Anti P2x7r C Terminal, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p2x7r c terminal/product/Alomone Labs
    Average 86 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti p2x7r c terminal - by Bioz Stars, 2024-06
    86/100 stars

    Images

    1) Product Images from "The NFATc1/P2X7 receptor relationship in human intervertebral disc cells"

    Article Title: The NFATc1/P2X7 receptor relationship in human intervertebral disc cells

    Journal: Frontiers in Cell and Developmental Biology

    doi: 10.3389/fcell.2024.1368318

    Overexpression of NFATc1 upregulates P2X7R expression in human IVD cells. IVD cells were transfected with 100 ng or 200 ng of pRSV-NFATc1/A expression vector (NFATc1) for 16 h and treated with PMA (20 nM) and ionomycin (1 μM) for 3 h. Cells were collected and subjected to RNA and protein analysis. mRNA levels of NFATc1 and P2X7R were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.01; ** p < 0.0001. Protein levels of NFATc1 and P2X7R were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.05. Scale bar = 20 μm.
    Figure Legend Snippet: Overexpression of NFATc1 upregulates P2X7R expression in human IVD cells. IVD cells were transfected with 100 ng or 200 ng of pRSV-NFATc1/A expression vector (NFATc1) for 16 h and treated with PMA (20 nM) and ionomycin (1 μM) for 3 h. Cells were collected and subjected to RNA and protein analysis. mRNA levels of NFATc1 and P2X7R were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.01; ** p < 0.0001. Protein levels of NFATc1 and P2X7R were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.05. Scale bar = 20 μm.

    Techniques Used: Over Expression, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunocytochemistry, Immunostaining, Software

    Association between P2X7R and NFATc1. (A) Immunofluorescence detection of P2X7R and NFATc1. The IVD cells were co-labeled with anti-P2X7R C-terminal antibody (green) and anti-NFATc1 antibody (red). Nuclei were counterstained with DAPI (blue). Representative images together with high-magnification pictures are reported (the nucleus chosen for magnification is indicated with a white arrowhead). Merge images represent an overlay of the two channels where colocalization is indicated by a color change (yellow). The graph indicates the fluorescence intensity profile along the white arrow. The blue, red, and green lines correspond to signals of DNA, NFATc1, and P2X7R, respectively. Scale bar = 10 μm; high-magnification pictures, scale bar = 2 μm. (B) CoIP assay to evaluate the interaction between NFATc1 and P2X7R. Immunoprecipitations of NFATc1 followed by immunoblot analyses with anti-NFATc1 and anti-P2X7R antibodies were performed in IVD cells derived from four patients with the same grade of degeneration pooled together. The negative control of immunoprecipitation (normal mouse IgG) was also included. (C) PLA of P2X7R and NFATc1 interactions performed in IVD cells using primary antibody against P2X7R and NFATc1. Detection of P2X7R was performed by immunofluorescence (green). Interactions between P2X7R and NFATc1 are revealed as red dots. Nuclei were counterstained with DAPI (blue). Quantification of positive PLA signals per cell is reported as a dot plot with mean (±SD, n = 3). PLA dot counts per cytoplasm and nucleus are reported separately. Data analyzed were based on an average of 100 cells. Scale bar = 10 μm; high-magnification pictures, scale bar = 5 μm. (D) Immunofluorescence detection of P2X7R and lamin A/C. The IVD cells were co-labeled with anti-P2X7R C-terminal antibody (green) and anti-lamin A/C antibody (red). Nuclei were counterstained with DAPI (blue). Representative images together with high-magnification pictures are reported (the nucleus chosen for magnification is indicated with a white arrowhead). Merge images represent an overlay of the two channels where colocalization is indicated by a color change (yellow). The graph indicates the fluorescence intensity profile along the white arrow. The blue, red, and green lines correspond to signals of DNA, lamin A/C, and P2X7R, respectively. Scale bar = 10 μm; high-magnification pictures, scale bar = 2 μm. (E) PLA of P2X7R and lamin A/C interactions performed in IVD cells using a primary antibody against P2X7R and lamin A/C. Detection of lamin A/C was performed by immunofluorescence (green). Interactions between P2X7R and lamin A/C are revealed as red dots. Nuclei were counterstained with DAPI (blue). Quantification of positive PLA signals per nucleus is reported as dot plot with mean (±SD, n = 3). Data analyzed were based on an average of 100 cells. Scale bar = 10 μm; high-magnification pictures, scale bar = 3 μm.
    Figure Legend Snippet: Association between P2X7R and NFATc1. (A) Immunofluorescence detection of P2X7R and NFATc1. The IVD cells were co-labeled with anti-P2X7R C-terminal antibody (green) and anti-NFATc1 antibody (red). Nuclei were counterstained with DAPI (blue). Representative images together with high-magnification pictures are reported (the nucleus chosen for magnification is indicated with a white arrowhead). Merge images represent an overlay of the two channels where colocalization is indicated by a color change (yellow). The graph indicates the fluorescence intensity profile along the white arrow. The blue, red, and green lines correspond to signals of DNA, NFATc1, and P2X7R, respectively. Scale bar = 10 μm; high-magnification pictures, scale bar = 2 μm. (B) CoIP assay to evaluate the interaction between NFATc1 and P2X7R. Immunoprecipitations of NFATc1 followed by immunoblot analyses with anti-NFATc1 and anti-P2X7R antibodies were performed in IVD cells derived from four patients with the same grade of degeneration pooled together. The negative control of immunoprecipitation (normal mouse IgG) was also included. (C) PLA of P2X7R and NFATc1 interactions performed in IVD cells using primary antibody against P2X7R and NFATc1. Detection of P2X7R was performed by immunofluorescence (green). Interactions between P2X7R and NFATc1 are revealed as red dots. Nuclei were counterstained with DAPI (blue). Quantification of positive PLA signals per cell is reported as a dot plot with mean (±SD, n = 3). PLA dot counts per cytoplasm and nucleus are reported separately. Data analyzed were based on an average of 100 cells. Scale bar = 10 μm; high-magnification pictures, scale bar = 5 μm. (D) Immunofluorescence detection of P2X7R and lamin A/C. The IVD cells were co-labeled with anti-P2X7R C-terminal antibody (green) and anti-lamin A/C antibody (red). Nuclei were counterstained with DAPI (blue). Representative images together with high-magnification pictures are reported (the nucleus chosen for magnification is indicated with a white arrowhead). Merge images represent an overlay of the two channels where colocalization is indicated by a color change (yellow). The graph indicates the fluorescence intensity profile along the white arrow. The blue, red, and green lines correspond to signals of DNA, lamin A/C, and P2X7R, respectively. Scale bar = 10 μm; high-magnification pictures, scale bar = 2 μm. (E) PLA of P2X7R and lamin A/C interactions performed in IVD cells using a primary antibody against P2X7R and lamin A/C. Detection of lamin A/C was performed by immunofluorescence (green). Interactions between P2X7R and lamin A/C are revealed as red dots. Nuclei were counterstained with DAPI (blue). Quantification of positive PLA signals per nucleus is reported as dot plot with mean (±SD, n = 3). Data analyzed were based on an average of 100 cells. Scale bar = 10 μm; high-magnification pictures, scale bar = 3 μm.

    Techniques Used: Immunofluorescence, Labeling, Fluorescence, Co-Immunoprecipitation Assay, Western Blot, Derivative Assay, Negative Control, Immunoprecipitation

    Regulation of NFATc1 and P2X7R expression under hypoxia. (A) HIF-1α expression was analyzed by immunocytochemistry in IVD cells during the de-differentiation process from P0 to P2 and in IVD cells cultured in the absence (untreated) or presence of CoCl 2 for 24 h. Representative photomicrographs are reported. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to P0 cells or untreated cells. Data are presented as the mean ± SD (n = 4). * p < 0.0001 P2 vs P0; ** p < 0.05 CoCl 2 vs untreated. Scale bar = 20 μm. (B, C) IVD cells were cultured in the absence (untreated) or presence of CoCl 2 for 24 h. Cells were then collected and subjected to RNA (B) and protein (C) analyses. mRNA levels of P2X7R and NFATc1 were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.0001 CoCl 2 vs untreated. Protein levels were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.0001. Scale bar = 20 μm. (D) ChIP analysis was performed in IVD cells cultured in the absence (untreated) or presence of CoCl 2 . The recognition motifs of HIF-1α from the JASPAR database and the positioning of potential hypoxia-responsive elements (HREs) within the P2RX7 (−2000/+32) promoter region are reported (HREs, triangle; NFATc1 potential binding sites, gray rectangles). The positions of specific primers used for qPCR amplification are also reported. Results of qPCR were analyzed using the 2 −ΔΔCt method, normalized for the input signal, and presented as fold enrichment with respect to the IgG negative control. Data are expressed as the mean ± SD (n = 2).
    Figure Legend Snippet: Regulation of NFATc1 and P2X7R expression under hypoxia. (A) HIF-1α expression was analyzed by immunocytochemistry in IVD cells during the de-differentiation process from P0 to P2 and in IVD cells cultured in the absence (untreated) or presence of CoCl 2 for 24 h. Representative photomicrographs are reported. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to P0 cells or untreated cells. Data are presented as the mean ± SD (n = 4). * p < 0.0001 P2 vs P0; ** p < 0.05 CoCl 2 vs untreated. Scale bar = 20 μm. (B, C) IVD cells were cultured in the absence (untreated) or presence of CoCl 2 for 24 h. Cells were then collected and subjected to RNA (B) and protein (C) analyses. mRNA levels of P2X7R and NFATc1 were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.0001 CoCl 2 vs untreated. Protein levels were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.0001. Scale bar = 20 μm. (D) ChIP analysis was performed in IVD cells cultured in the absence (untreated) or presence of CoCl 2 . The recognition motifs of HIF-1α from the JASPAR database and the positioning of potential hypoxia-responsive elements (HREs) within the P2RX7 (−2000/+32) promoter region are reported (HREs, triangle; NFATc1 potential binding sites, gray rectangles). The positions of specific primers used for qPCR amplification are also reported. Results of qPCR were analyzed using the 2 −ΔΔCt method, normalized for the input signal, and presented as fold enrichment with respect to the IgG negative control. Data are expressed as the mean ± SD (n = 2).

    Techniques Used: Expressing, Immunocytochemistry, Cell Culture, Software, Quantitative RT-PCR, Immunostaining, Binding Assay, Amplification, Negative Control

    rabbit anti p2x7r  (Alomone Labs)


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    Structured Review

    Alomone Labs rabbit anti p2x7r
    <t>P2X7R</t> expression in blood of patients with GBS and healthy controls. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood samples, and P2X7R expression was examined using flow cytometry and qRT-PCR. (A) P2X7R mRNA levels were elevated in the PBMCs collected from patients with GBS compared to those in PBMCs collected from the healthy controls ( n = 8 per group). (B-D) P2X7R expression increased on CD4 + T cells in the PBMCs collected from patients with GBS, but no significant increase was noted on CD8 + T cells or monocytes (CD14 + ) compared to healthy controls ( n = 7 per group). HC, healthy control; GBS, Guillain–Barre Syndrome. * p < 0.05, ** p < 0.01
    Rabbit Anti P2x7r, 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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    Images

    1) Product Images from "P2X7 receptor antagonists modulate experimental autoimmune neuritis via regulation of NLRP3 inflammasome activation and Th17 and Th1 cell differentiation"

    Article Title: P2X7 receptor antagonists modulate experimental autoimmune neuritis via regulation of NLRP3 inflammasome activation and Th17 and Th1 cell differentiation

    Journal: Journal of Neuroinflammation

    doi: 10.1186/s12974-024-03057-z

    P2X7R expression in blood of patients with GBS and healthy controls. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood samples, and P2X7R expression was examined using flow cytometry and qRT-PCR. (A) P2X7R mRNA levels were elevated in the PBMCs collected from patients with GBS compared to those in PBMCs collected from the healthy controls ( n = 8 per group). (B-D) P2X7R expression increased on CD4 + T cells in the PBMCs collected from patients with GBS, but no significant increase was noted on CD8 + T cells or monocytes (CD14 + ) compared to healthy controls ( n = 7 per group). HC, healthy control; GBS, Guillain–Barre Syndrome. * p < 0.05, ** p < 0.01
    Figure Legend Snippet: P2X7R expression in blood of patients with GBS and healthy controls. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood samples, and P2X7R expression was examined using flow cytometry and qRT-PCR. (A) P2X7R mRNA levels were elevated in the PBMCs collected from patients with GBS compared to those in PBMCs collected from the healthy controls ( n = 8 per group). (B-D) P2X7R expression increased on CD4 + T cells in the PBMCs collected from patients with GBS, but no significant increase was noted on CD8 + T cells or monocytes (CD14 + ) compared to healthy controls ( n = 7 per group). HC, healthy control; GBS, Guillain–Barre Syndrome. * p < 0.05, ** p < 0.01

    Techniques Used: Expressing, Isolation, Flow Cytometry, Quantitative RT-PCR

    P2X7R expression was significantly elevated in CD4 + T cells in EAN rats. P2X7R expression in T cells and macrophages in control (unimmunized group) and EAN rats at day 18. (A-B) Fluorescence photomicrographs showing P2X7R expression and quantification in CD4 + T cells, CD8 + T cells and macrophages (CD68 + ) in the sciatic nerves of EAN and control rats. Scale bars, 20 μm. (C) Graph showing the number of P2X7R-positive cells in sciatic nerve sections obtained from control and EAN rats. (D-E) Flow cytometry analysis of P2X7R expression and quantification on CD4 + T cells, CD8 + T cells and macrophages in the spleen of EAN and control rats. Experiments were performed in triplicate ( n = 6 per replicate) with similar results. * p < 0.05, ** p < 0.01
    Figure Legend Snippet: P2X7R expression was significantly elevated in CD4 + T cells in EAN rats. P2X7R expression in T cells and macrophages in control (unimmunized group) and EAN rats at day 18. (A-B) Fluorescence photomicrographs showing P2X7R expression and quantification in CD4 + T cells, CD8 + T cells and macrophages (CD68 + ) in the sciatic nerves of EAN and control rats. Scale bars, 20 μm. (C) Graph showing the number of P2X7R-positive cells in sciatic nerve sections obtained from control and EAN rats. (D-E) Flow cytometry analysis of P2X7R expression and quantification on CD4 + T cells, CD8 + T cells and macrophages in the spleen of EAN and control rats. Experiments were performed in triplicate ( n = 6 per replicate) with similar results. * p < 0.05, ** p < 0.01

    Techniques Used: Expressing, Fluorescence, Flow Cytometry

    BBG treatment suppressed the P2X7R/NLRP3 pathway in CD4 + T cells in EAN. Proteins were obtained from sciatic nerves at the peak of EAN and subjected to western blot analysis and quantification. NLRP3 expression in immune cells was analyzed through flow cytometry and immunofluorescence. (A) Representative western blots of P2X7R, NLRP3, IL-1β and Caspase-1 protein expression in sciatic nerve. (B-E) Protein levels were quantified via densitometric analysis of immunoreactive bands in western blots using ImageJ software and were normalized to that of GAPDH. (F-G) Flow cytometry analysis and NLRP3 quantification on CD4 + T cells in splenic MNCs of control (unimmunized group), vehicle and BBG-treated rats. (H) Immunofluorescence photomicrographs of NLRP3 expression in CD4 + T cells in the sciatic nerves of control, vehicle, and BBG-treated EAN rats. (I) Number of NLRP3-positive cells in sciatic nerve sections obtained from control rats, vehicle and BBG-treated EAN rats. (J) NLRP3 quantification on CD4 + T cells. Scale bars, 20 μm. Experiments were performed in triplicate ( n = 6 per replicate) with similar results. Control group, unimmunized group; BBG-P, preventative BBG group; BBG-T, therapeutic BBG group. * p < 0.05, ** p < 0.01
    Figure Legend Snippet: BBG treatment suppressed the P2X7R/NLRP3 pathway in CD4 + T cells in EAN. Proteins were obtained from sciatic nerves at the peak of EAN and subjected to western blot analysis and quantification. NLRP3 expression in immune cells was analyzed through flow cytometry and immunofluorescence. (A) Representative western blots of P2X7R, NLRP3, IL-1β and Caspase-1 protein expression in sciatic nerve. (B-E) Protein levels were quantified via densitometric analysis of immunoreactive bands in western blots using ImageJ software and were normalized to that of GAPDH. (F-G) Flow cytometry analysis and NLRP3 quantification on CD4 + T cells in splenic MNCs of control (unimmunized group), vehicle and BBG-treated rats. (H) Immunofluorescence photomicrographs of NLRP3 expression in CD4 + T cells in the sciatic nerves of control, vehicle, and BBG-treated EAN rats. (I) Number of NLRP3-positive cells in sciatic nerve sections obtained from control rats, vehicle and BBG-treated EAN rats. (J) NLRP3 quantification on CD4 + T cells. Scale bars, 20 μm. Experiments were performed in triplicate ( n = 6 per replicate) with similar results. Control group, unimmunized group; BBG-P, preventative BBG group; BBG-T, therapeutic BBG group. * p < 0.05, ** p < 0.01

    Techniques Used: Western Blot, Expressing, Flow Cytometry, Immunofluorescence, Software

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    Alomone Labs antibodies against p2x7r
    Immunohistochemical (IHC) staining and IHC-quantitative analysis of <t>P2X7R</t> and P2X4R in the SMGs of CON and DM groups: ( a – d ) IHC images of P2X7R expression in the CON11w, DM11w, CON13w, and DM13w groups. ( e – h ) IHC images of P2X4R in the CON11w, DM11w, CON13w, and DM13w groups. ( i – l ) IHC images of negative control (NC) in the CON11w, DM11w, CON13w, and DM13w groups. Scale bars = 50 μm. ( m ) The P2X7R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. ( n ) The P2X4R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. The data were presented as the means ± SEM (* p < 0.05, *** p < 0.001 and ns not significant).
    Antibodies Against P2x7r, 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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    Alomone Labs anti p2x7r
    Immunohistochemical (IHC) staining and IHC-quantitative analysis of <t>P2X7R</t> and P2X4R in the SMGs of CON and DM groups: ( a – d ) IHC images of P2X7R expression in the CON11w, DM11w, CON13w, and DM13w groups. ( e – h ) IHC images of P2X4R in the CON11w, DM11w, CON13w, and DM13w groups. ( i – l ) IHC images of negative control (NC) in the CON11w, DM11w, CON13w, and DM13w groups. Scale bars = 50 μm. ( m ) The P2X7R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. ( n ) The P2X4R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. The data were presented as the means ± SEM (* p < 0.05, *** p < 0.001 and ns not significant).
    Anti P2x7r, 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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    86
    Alomone Labs antibodies for p2x7r
    Immunohistochemical (IHC) staining and IHC-quantitative analysis of <t>P2X7R</t> and P2X4R in the SMGs of CON and DM groups: ( a – d ) IHC images of P2X7R expression in the CON11w, DM11w, CON13w, and DM13w groups. ( e – h ) IHC images of P2X4R in the CON11w, DM11w, CON13w, and DM13w groups. ( i – l ) IHC images of negative control (NC) in the CON11w, DM11w, CON13w, and DM13w groups. Scale bars = 50 μm. ( m ) The P2X7R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. ( n ) The P2X4R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. The data were presented as the means ± SEM (* p < 0.05, *** p < 0.001 and ns not significant).
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    Alomone Labs p2x7r
    <t> P2X7R </t> SNPs mutations in PDAC patients and controls
    P2x7r, 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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    Alomone Labs anti p2x7r c terminal
    Overexpression of NFATc1 upregulates <t>P2X7R</t> expression in human IVD cells. IVD cells were transfected with 100 ng or 200 ng of pRSV-NFATc1/A expression vector (NFATc1) for 16 h and treated with PMA (20 nM) and ionomycin (1 μM) for 3 h. Cells were collected and subjected to RNA and protein analysis. mRNA levels of NFATc1 and P2X7R were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.01; ** p < 0.0001. Protein levels of NFATc1 and P2X7R were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.05. Scale bar = 20 μm.
    Anti P2x7r C Terminal, 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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    86
    Alomone Labs rabbit anti p2x7r
    <t>P2X7R</t> expression in blood of patients with GBS and healthy controls. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood samples, and P2X7R expression was examined using flow cytometry and qRT-PCR. (A) P2X7R mRNA levels were elevated in the PBMCs collected from patients with GBS compared to those in PBMCs collected from the healthy controls ( n = 8 per group). (B-D) P2X7R expression increased on CD4 + T cells in the PBMCs collected from patients with GBS, but no significant increase was noted on CD8 + T cells or monocytes (CD14 + ) compared to healthy controls ( n = 7 per group). HC, healthy control; GBS, Guillain–Barre Syndrome. * p < 0.05, ** p < 0.01
    Rabbit Anti P2x7r, 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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    Image Search Results


    Immunohistochemical (IHC) staining and IHC-quantitative analysis of P2X7R and P2X4R in the SMGs of CON and DM groups: ( a – d ) IHC images of P2X7R expression in the CON11w, DM11w, CON13w, and DM13w groups. ( e – h ) IHC images of P2X4R in the CON11w, DM11w, CON13w, and DM13w groups. ( i – l ) IHC images of negative control (NC) in the CON11w, DM11w, CON13w, and DM13w groups. Scale bars = 50 μm. ( m ) The P2X7R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. ( n ) The P2X4R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. The data were presented as the means ± SEM (* p < 0.05, *** p < 0.001 and ns not significant).

    Journal: Scientific Reports

    Article Title: P2X7R and P2X4R expression of mice submandibular gland in high-fat diet/streptozotocin-induced type 2 diabetes

    doi: 10.1038/s41598-024-60519-3

    Figure Lengend Snippet: Immunohistochemical (IHC) staining and IHC-quantitative analysis of P2X7R and P2X4R in the SMGs of CON and DM groups: ( a – d ) IHC images of P2X7R expression in the CON11w, DM11w, CON13w, and DM13w groups. ( e – h ) IHC images of P2X4R in the CON11w, DM11w, CON13w, and DM13w groups. ( i – l ) IHC images of negative control (NC) in the CON11w, DM11w, CON13w, and DM13w groups. Scale bars = 50 μm. ( m ) The P2X7R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. ( n ) The P2X4R area in the CON11w, DM11w, CON13w, and DM13w groups measured from 20 random fields of 300 × 300 pixels (0.05 × 0.05 mm) of two sections from each mouse. Dots in the graph represent 240 images in each group. The data were presented as the means ± SEM (* p < 0.05, *** p < 0.001 and ns not significant).

    Article Snippet: The sections were then incubated with primary antibodies against P2X7R (#APR-004; Alomone Laboratories, Jerusalem, Israel) and P2X4R (#APR-002; Alomone Laboratories, Jerusalem, Israel) in PBS (diluted for P2X4R = 1:200, P2X7R = 1:500) overnight at 4 °C.

    Techniques: Immunohistochemical staining, Immunohistochemistry, Expressing, Negative Control

    RT-qPCR analysis of P2X7R and P2X4R in the SMG of the CON and DM groups; ( a ) P2X7R mRNA expression in the CON11w, DM11w, CON13w, and DM13w groups (n = 6 each). ( b ) P2X4R mRNA expression in the CON11w, DM11w, CON13w, and DM13w groups (n = 6 each). The data were presented as the SEM (* p < 0.05, ** p < 0.01 and ns not significant).

    Journal: Scientific Reports

    Article Title: P2X7R and P2X4R expression of mice submandibular gland in high-fat diet/streptozotocin-induced type 2 diabetes

    doi: 10.1038/s41598-024-60519-3

    Figure Lengend Snippet: RT-qPCR analysis of P2X7R and P2X4R in the SMG of the CON and DM groups; ( a ) P2X7R mRNA expression in the CON11w, DM11w, CON13w, and DM13w groups (n = 6 each). ( b ) P2X4R mRNA expression in the CON11w, DM11w, CON13w, and DM13w groups (n = 6 each). The data were presented as the SEM (* p < 0.05, ** p < 0.01 and ns not significant).

    Article Snippet: The sections were then incubated with primary antibodies against P2X7R (#APR-004; Alomone Laboratories, Jerusalem, Israel) and P2X4R (#APR-002; Alomone Laboratories, Jerusalem, Israel) in PBS (diluted for P2X4R = 1:200, P2X7R = 1:500) overnight at 4 °C.

    Techniques: Quantitative RT-PCR, Expressing

    Primer sequences used in RT-qPCR process.

    Journal: Scientific Reports

    Article Title: P2X7R and P2X4R expression of mice submandibular gland in high-fat diet/streptozotocin-induced type 2 diabetes

    doi: 10.1038/s41598-024-60519-3

    Figure Lengend Snippet: Primer sequences used in RT-qPCR process.

    Article Snippet: The sections were then incubated with primary antibodies against P2X7R (#APR-004; Alomone Laboratories, Jerusalem, Israel) and P2X4R (#APR-002; Alomone Laboratories, Jerusalem, Israel) in PBS (diluted for P2X4R = 1:200, P2X7R = 1:500) overnight at 4 °C.

    Techniques: Sequencing

     P2X7R  SNPs mutations in PDAC patients and controls

    Journal: Cancer Cell International

    Article Title: Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer

    doi: 10.1186/s12935-024-03339-9

    Figure Lengend Snippet: P2X7R SNPs mutations in PDAC patients and controls

    Article Snippet: Primary antibodies were against the P2X7R (APR-004, Alomone Labs, Jerusalem, IL, RRID: AB_2040068), GFP (SC-8334, Santa Cruz, Tilst, DK, RRID:AB_641123), β-Actin (Sc-47778, Santa Cruz, Tilst, DK, RRID: AB_626632) and secondary antibody was HRP-conjugated (EZ-ECL-Biological Industries, Fredensborg, DK) and visualized with Fusion FX (Vilber Lourmat, Eberhardzell, DE).

    Techniques:

    Cell morphology and P2X7R expression in PANC-1 and PSC cells. a Representative fluorescence and transmission images of cells expressing P2X7R + GFP variants. Bars are 50 µm. b Representative WB on whole cell lysate showing P2X7R expression in transfected and non-transfected (NT) cells. GFP and β-actin are also shown as transfection and loading control, respectively

    Journal: Cancer Cell International

    Article Title: Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer

    doi: 10.1186/s12935-024-03339-9

    Figure Lengend Snippet: Cell morphology and P2X7R expression in PANC-1 and PSC cells. a Representative fluorescence and transmission images of cells expressing P2X7R + GFP variants. Bars are 50 µm. b Representative WB on whole cell lysate showing P2X7R expression in transfected and non-transfected (NT) cells. GFP and β-actin are also shown as transfection and loading control, respectively

    Article Snippet: Primary antibodies were against the P2X7R (APR-004, Alomone Labs, Jerusalem, IL, RRID: AB_2040068), GFP (SC-8334, Santa Cruz, Tilst, DK, RRID:AB_641123), β-Actin (Sc-47778, Santa Cruz, Tilst, DK, RRID: AB_626632) and secondary antibody was HRP-conjugated (EZ-ECL-Biological Industries, Fredensborg, DK) and visualized with Fusion FX (Vilber Lourmat, Eberhardzell, DE).

    Techniques: Expressing, Fluorescence, Transmission Assay, Transfection

    Effect of P2X7R SNPs on calcium transients in PANC-1, PSCs and HEK293. a Representative recordings of Ca 2+ signals (Fura-2 ratio) in P2X7R + GFP PANC-1 and PSCs (top and bottom panels) transfected with WT (blue), Gly150Arg (orange) and Arg276His (magenta) receptor. The graphs show the response after stimulation with BzATP 100 µM (grey arrow) and the positive control ionomycin 1 µM (green arrow). b Area under the curve (AUC) calculated for 600 s (PANC-1), 400 s (PSCs) and 160 s (HEK293) after the response starts. c Change in Fura-2 (ΔFura-2) between the basal and the maximum peak response to the agonist. Each point represents the change of Fura-2 in a single cell. Comparison between two variants have been evaluated with the non-parametric Mann–Whitney test and the p values are reported in the graphs. Each graph in ( b , c ) shows responses of cells in 3–4 independent experiments and medians and the interquartile range

    Journal: Cancer Cell International

    Article Title: Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer

    doi: 10.1186/s12935-024-03339-9

    Figure Lengend Snippet: Effect of P2X7R SNPs on calcium transients in PANC-1, PSCs and HEK293. a Representative recordings of Ca 2+ signals (Fura-2 ratio) in P2X7R + GFP PANC-1 and PSCs (top and bottom panels) transfected with WT (blue), Gly150Arg (orange) and Arg276His (magenta) receptor. The graphs show the response after stimulation with BzATP 100 µM (grey arrow) and the positive control ionomycin 1 µM (green arrow). b Area under the curve (AUC) calculated for 600 s (PANC-1), 400 s (PSCs) and 160 s (HEK293) after the response starts. c Change in Fura-2 (ΔFura-2) between the basal and the maximum peak response to the agonist. Each point represents the change of Fura-2 in a single cell. Comparison between two variants have been evaluated with the non-parametric Mann–Whitney test and the p values are reported in the graphs. Each graph in ( b , c ) shows responses of cells in 3–4 independent experiments and medians and the interquartile range

    Article Snippet: Primary antibodies were against the P2X7R (APR-004, Alomone Labs, Jerusalem, IL, RRID: AB_2040068), GFP (SC-8334, Santa Cruz, Tilst, DK, RRID:AB_641123), β-Actin (Sc-47778, Santa Cruz, Tilst, DK, RRID: AB_626632) and secondary antibody was HRP-conjugated (EZ-ECL-Biological Industries, Fredensborg, DK) and visualized with Fusion FX (Vilber Lourmat, Eberhardzell, DE).

    Techniques: Transfection, Positive Control, Comparison, MANN-WHITNEY

    Reduced ATP-induced dye uptake in PANC-1, PSCs and HEK293 cells expressing Gly150Arg and Arg276His. a Original single-cell traces of the ATP-induced YO-PRO-1 uptake in P2X7R + GFP PANC-1 (top) and PSCs (bottom) expressing WT (blue), Gly150Arg (orange) and Arg276His (magenta) receptors. YO-PRO-1 was added after 25 min (beige arrow) prior to stimulation with 5 mM ATP (green arrow). b Comparison of AUC of P2X7R + GFP expressing the different P2X7R variants. Statistical significance was evaluated with the non-parametric Mann–Whitney test and the p values are reported in the graphs. Graphs in ( b ) and ( c ) include results from single cells obtained in 3–4 independent experiments

    Journal: Cancer Cell International

    Article Title: Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer

    doi: 10.1186/s12935-024-03339-9

    Figure Lengend Snippet: Reduced ATP-induced dye uptake in PANC-1, PSCs and HEK293 cells expressing Gly150Arg and Arg276His. a Original single-cell traces of the ATP-induced YO-PRO-1 uptake in P2X7R + GFP PANC-1 (top) and PSCs (bottom) expressing WT (blue), Gly150Arg (orange) and Arg276His (magenta) receptors. YO-PRO-1 was added after 25 min (beige arrow) prior to stimulation with 5 mM ATP (green arrow). b Comparison of AUC of P2X7R + GFP expressing the different P2X7R variants. Statistical significance was evaluated with the non-parametric Mann–Whitney test and the p values are reported in the graphs. Graphs in ( b ) and ( c ) include results from single cells obtained in 3–4 independent experiments

    Article Snippet: Primary antibodies were against the P2X7R (APR-004, Alomone Labs, Jerusalem, IL, RRID: AB_2040068), GFP (SC-8334, Santa Cruz, Tilst, DK, RRID:AB_641123), β-Actin (Sc-47778, Santa Cruz, Tilst, DK, RRID: AB_626632) and secondary antibody was HRP-conjugated (EZ-ECL-Biological Industries, Fredensborg, DK) and visualized with Fusion FX (Vilber Lourmat, Eberhardzell, DE).

    Techniques: Expressing, Comparison, MANN-WHITNEY

    Effects of the P2X7R variants on cell-specific functions: cytokine release from PSCs ( a – d ) and migration of PANC-1 cells ( e ). a – d IL-6, IL-1β, IL-8 and TNF-α released in the supernatant of PSCs expressing WT (blue), Gly150Arg (orange) and Arg276His (magenta) receptors. Stimulation with BzATP 100 µM and inhibition with AZ10606120 (AZ) 10 µM and A438079 (A43) 10 µM are compared to the respective unstimulated controls cells (CTR). The data are shown as the mean ± SEM for IL-6, IL-1β and TNF-α, and median ± interquartile range for IL-8, of n = 9. e Migration of P2X7R + GFP PANC-1 expressing P2X7R variants . Significance has been evaluated as followed: IL-6, IL-1β and TNF-α statistics have been performed with one sample t test, and reported as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; IL-8 statistics have been performed with one-sample Wilcoxon signed rank test and reported as *p < 0.05; **p < 0.01. Migration has been analyzed with the non-parametric Mann–Whitney test and the p values are reported in the graph

    Journal: Cancer Cell International

    Article Title: Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer

    doi: 10.1186/s12935-024-03339-9

    Figure Lengend Snippet: Effects of the P2X7R variants on cell-specific functions: cytokine release from PSCs ( a – d ) and migration of PANC-1 cells ( e ). a – d IL-6, IL-1β, IL-8 and TNF-α released in the supernatant of PSCs expressing WT (blue), Gly150Arg (orange) and Arg276His (magenta) receptors. Stimulation with BzATP 100 µM and inhibition with AZ10606120 (AZ) 10 µM and A438079 (A43) 10 µM are compared to the respective unstimulated controls cells (CTR). The data are shown as the mean ± SEM for IL-6, IL-1β and TNF-α, and median ± interquartile range for IL-8, of n = 9. e Migration of P2X7R + GFP PANC-1 expressing P2X7R variants . Significance has been evaluated as followed: IL-6, IL-1β and TNF-α statistics have been performed with one sample t test, and reported as *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001; IL-8 statistics have been performed with one-sample Wilcoxon signed rank test and reported as *p < 0.05; **p < 0.01. Migration has been analyzed with the non-parametric Mann–Whitney test and the p values are reported in the graph

    Article Snippet: Primary antibodies were against the P2X7R (APR-004, Alomone Labs, Jerusalem, IL, RRID: AB_2040068), GFP (SC-8334, Santa Cruz, Tilst, DK, RRID:AB_641123), β-Actin (Sc-47778, Santa Cruz, Tilst, DK, RRID: AB_626632) and secondary antibody was HRP-conjugated (EZ-ECL-Biological Industries, Fredensborg, DK) and visualized with Fusion FX (Vilber Lourmat, Eberhardzell, DE).

    Techniques: Migration, Expressing, Inhibition, MANN-WHITNEY

    Overexpression of NFATc1 upregulates P2X7R expression in human IVD cells. IVD cells were transfected with 100 ng or 200 ng of pRSV-NFATc1/A expression vector (NFATc1) for 16 h and treated with PMA (20 nM) and ionomycin (1 μM) for 3 h. Cells were collected and subjected to RNA and protein analysis. mRNA levels of NFATc1 and P2X7R were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.01; ** p < 0.0001. Protein levels of NFATc1 and P2X7R were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.05. Scale bar = 20 μm.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: The NFATc1/P2X7 receptor relationship in human intervertebral disc cells

    doi: 10.3389/fcell.2024.1368318

    Figure Lengend Snippet: Overexpression of NFATc1 upregulates P2X7R expression in human IVD cells. IVD cells were transfected with 100 ng or 200 ng of pRSV-NFATc1/A expression vector (NFATc1) for 16 h and treated with PMA (20 nM) and ionomycin (1 μM) for 3 h. Cells were collected and subjected to RNA and protein analysis. mRNA levels of NFATc1 and P2X7R were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.01; ** p < 0.0001. Protein levels of NFATc1 and P2X7R were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.05. Scale bar = 20 μm.

    Article Snippet: The primary antibodies against SOX9 (# sc-20095), aggrecan (ACAN, # sc-33695), HIF-1α (H1α67, # sc-53546), NFATc1 (7A6, # sc-7294; H-10, # sc-1783; H-110, # sc-13033), lamin A/C (E-1, # sc-376248), and normal mouse IgG 1 (# sc-3877) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, United States), while anti-collagen type II α1 chain (COL2a1; # ab3092) antibody was purchased from Abcam (Cambridge, United Kingdom), anti-HIF-1α (H1 alpha67, # NB100–134) was purchased from Novus Biologicals (Centennial, CO, United States), anti-P2X7R C-terminal (# APR-004) was purchased from Alomone Labs (Jerusalem, Israel), and anti-P2X7R C-terminal (#P8232) and anti-P2X7R extracellular loop (#P9122) were purchased from Sigma-Aldrich, Merck KGaA (Milan, Italy).

    Techniques: Over Expression, Expressing, Transfection, Plasmid Preparation, Quantitative RT-PCR, Immunocytochemistry, Immunostaining, Software

    Association between P2X7R and NFATc1. (A) Immunofluorescence detection of P2X7R and NFATc1. The IVD cells were co-labeled with anti-P2X7R C-terminal antibody (green) and anti-NFATc1 antibody (red). Nuclei were counterstained with DAPI (blue). Representative images together with high-magnification pictures are reported (the nucleus chosen for magnification is indicated with a white arrowhead). Merge images represent an overlay of the two channels where colocalization is indicated by a color change (yellow). The graph indicates the fluorescence intensity profile along the white arrow. The blue, red, and green lines correspond to signals of DNA, NFATc1, and P2X7R, respectively. Scale bar = 10 μm; high-magnification pictures, scale bar = 2 μm. (B) CoIP assay to evaluate the interaction between NFATc1 and P2X7R. Immunoprecipitations of NFATc1 followed by immunoblot analyses with anti-NFATc1 and anti-P2X7R antibodies were performed in IVD cells derived from four patients with the same grade of degeneration pooled together. The negative control of immunoprecipitation (normal mouse IgG) was also included. (C) PLA of P2X7R and NFATc1 interactions performed in IVD cells using primary antibody against P2X7R and NFATc1. Detection of P2X7R was performed by immunofluorescence (green). Interactions between P2X7R and NFATc1 are revealed as red dots. Nuclei were counterstained with DAPI (blue). Quantification of positive PLA signals per cell is reported as a dot plot with mean (±SD, n = 3). PLA dot counts per cytoplasm and nucleus are reported separately. Data analyzed were based on an average of 100 cells. Scale bar = 10 μm; high-magnification pictures, scale bar = 5 μm. (D) Immunofluorescence detection of P2X7R and lamin A/C. The IVD cells were co-labeled with anti-P2X7R C-terminal antibody (green) and anti-lamin A/C antibody (red). Nuclei were counterstained with DAPI (blue). Representative images together with high-magnification pictures are reported (the nucleus chosen for magnification is indicated with a white arrowhead). Merge images represent an overlay of the two channels where colocalization is indicated by a color change (yellow). The graph indicates the fluorescence intensity profile along the white arrow. The blue, red, and green lines correspond to signals of DNA, lamin A/C, and P2X7R, respectively. Scale bar = 10 μm; high-magnification pictures, scale bar = 2 μm. (E) PLA of P2X7R and lamin A/C interactions performed in IVD cells using a primary antibody against P2X7R and lamin A/C. Detection of lamin A/C was performed by immunofluorescence (green). Interactions between P2X7R and lamin A/C are revealed as red dots. Nuclei were counterstained with DAPI (blue). Quantification of positive PLA signals per nucleus is reported as dot plot with mean (±SD, n = 3). Data analyzed were based on an average of 100 cells. Scale bar = 10 μm; high-magnification pictures, scale bar = 3 μm.

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: The NFATc1/P2X7 receptor relationship in human intervertebral disc cells

    doi: 10.3389/fcell.2024.1368318

    Figure Lengend Snippet: Association between P2X7R and NFATc1. (A) Immunofluorescence detection of P2X7R and NFATc1. The IVD cells were co-labeled with anti-P2X7R C-terminal antibody (green) and anti-NFATc1 antibody (red). Nuclei were counterstained with DAPI (blue). Representative images together with high-magnification pictures are reported (the nucleus chosen for magnification is indicated with a white arrowhead). Merge images represent an overlay of the two channels where colocalization is indicated by a color change (yellow). The graph indicates the fluorescence intensity profile along the white arrow. The blue, red, and green lines correspond to signals of DNA, NFATc1, and P2X7R, respectively. Scale bar = 10 μm; high-magnification pictures, scale bar = 2 μm. (B) CoIP assay to evaluate the interaction between NFATc1 and P2X7R. Immunoprecipitations of NFATc1 followed by immunoblot analyses with anti-NFATc1 and anti-P2X7R antibodies were performed in IVD cells derived from four patients with the same grade of degeneration pooled together. The negative control of immunoprecipitation (normal mouse IgG) was also included. (C) PLA of P2X7R and NFATc1 interactions performed in IVD cells using primary antibody against P2X7R and NFATc1. Detection of P2X7R was performed by immunofluorescence (green). Interactions between P2X7R and NFATc1 are revealed as red dots. Nuclei were counterstained with DAPI (blue). Quantification of positive PLA signals per cell is reported as a dot plot with mean (±SD, n = 3). PLA dot counts per cytoplasm and nucleus are reported separately. Data analyzed were based on an average of 100 cells. Scale bar = 10 μm; high-magnification pictures, scale bar = 5 μm. (D) Immunofluorescence detection of P2X7R and lamin A/C. The IVD cells were co-labeled with anti-P2X7R C-terminal antibody (green) and anti-lamin A/C antibody (red). Nuclei were counterstained with DAPI (blue). Representative images together with high-magnification pictures are reported (the nucleus chosen for magnification is indicated with a white arrowhead). Merge images represent an overlay of the two channels where colocalization is indicated by a color change (yellow). The graph indicates the fluorescence intensity profile along the white arrow. The blue, red, and green lines correspond to signals of DNA, lamin A/C, and P2X7R, respectively. Scale bar = 10 μm; high-magnification pictures, scale bar = 2 μm. (E) PLA of P2X7R and lamin A/C interactions performed in IVD cells using a primary antibody against P2X7R and lamin A/C. Detection of lamin A/C was performed by immunofluorescence (green). Interactions between P2X7R and lamin A/C are revealed as red dots. Nuclei were counterstained with DAPI (blue). Quantification of positive PLA signals per nucleus is reported as dot plot with mean (±SD, n = 3). Data analyzed were based on an average of 100 cells. Scale bar = 10 μm; high-magnification pictures, scale bar = 3 μm.

    Article Snippet: The primary antibodies against SOX9 (# sc-20095), aggrecan (ACAN, # sc-33695), HIF-1α (H1α67, # sc-53546), NFATc1 (7A6, # sc-7294; H-10, # sc-1783; H-110, # sc-13033), lamin A/C (E-1, # sc-376248), and normal mouse IgG 1 (# sc-3877) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, United States), while anti-collagen type II α1 chain (COL2a1; # ab3092) antibody was purchased from Abcam (Cambridge, United Kingdom), anti-HIF-1α (H1 alpha67, # NB100–134) was purchased from Novus Biologicals (Centennial, CO, United States), anti-P2X7R C-terminal (# APR-004) was purchased from Alomone Labs (Jerusalem, Israel), and anti-P2X7R C-terminal (#P8232) and anti-P2X7R extracellular loop (#P9122) were purchased from Sigma-Aldrich, Merck KGaA (Milan, Italy).

    Techniques: Immunofluorescence, Labeling, Fluorescence, Co-Immunoprecipitation Assay, Western Blot, Derivative Assay, Negative Control, Immunoprecipitation

    Regulation of NFATc1 and P2X7R expression under hypoxia. (A) HIF-1α expression was analyzed by immunocytochemistry in IVD cells during the de-differentiation process from P0 to P2 and in IVD cells cultured in the absence (untreated) or presence of CoCl 2 for 24 h. Representative photomicrographs are reported. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to P0 cells or untreated cells. Data are presented as the mean ± SD (n = 4). * p < 0.0001 P2 vs P0; ** p < 0.05 CoCl 2 vs untreated. Scale bar = 20 μm. (B, C) IVD cells were cultured in the absence (untreated) or presence of CoCl 2 for 24 h. Cells were then collected and subjected to RNA (B) and protein (C) analyses. mRNA levels of P2X7R and NFATc1 were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.0001 CoCl 2 vs untreated. Protein levels were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.0001. Scale bar = 20 μm. (D) ChIP analysis was performed in IVD cells cultured in the absence (untreated) or presence of CoCl 2 . The recognition motifs of HIF-1α from the JASPAR database and the positioning of potential hypoxia-responsive elements (HREs) within the P2RX7 (−2000/+32) promoter region are reported (HREs, triangle; NFATc1 potential binding sites, gray rectangles). The positions of specific primers used for qPCR amplification are also reported. Results of qPCR were analyzed using the 2 −ΔΔCt method, normalized for the input signal, and presented as fold enrichment with respect to the IgG negative control. Data are expressed as the mean ± SD (n = 2).

    Journal: Frontiers in Cell and Developmental Biology

    Article Title: The NFATc1/P2X7 receptor relationship in human intervertebral disc cells

    doi: 10.3389/fcell.2024.1368318

    Figure Lengend Snippet: Regulation of NFATc1 and P2X7R expression under hypoxia. (A) HIF-1α expression was analyzed by immunocytochemistry in IVD cells during the de-differentiation process from P0 to P2 and in IVD cells cultured in the absence (untreated) or presence of CoCl 2 for 24 h. Representative photomicrographs are reported. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to P0 cells or untreated cells. Data are presented as the mean ± SD (n = 4). * p < 0.0001 P2 vs P0; ** p < 0.05 CoCl 2 vs untreated. Scale bar = 20 μm. (B, C) IVD cells were cultured in the absence (untreated) or presence of CoCl 2 for 24 h. Cells were then collected and subjected to RNA (B) and protein (C) analyses. mRNA levels of P2X7R and NFATc1 were determined by real-time RT-PCR. RNA relative expression levels were normalized to untreated cells and expressed as fold change. Data represent means ± SD (n = 3). * p < 0.0001 CoCl 2 vs untreated. Protein levels were analyzed by immunocytochemistry, and representative optical photomicrographs of immunostaining are shown. Protein levels were quantified by densitometric analysis of immunocytochemical pictures using ImageJ software. Quantitative analysis of at least 10 fields per replicate was performed. Protein levels are expressed as fold change relative to untreated cells. Data are presented as the mean ± SD (n = 3). * p < 0.0001. Scale bar = 20 μm. (D) ChIP analysis was performed in IVD cells cultured in the absence (untreated) or presence of CoCl 2 . The recognition motifs of HIF-1α from the JASPAR database and the positioning of potential hypoxia-responsive elements (HREs) within the P2RX7 (−2000/+32) promoter region are reported (HREs, triangle; NFATc1 potential binding sites, gray rectangles). The positions of specific primers used for qPCR amplification are also reported. Results of qPCR were analyzed using the 2 −ΔΔCt method, normalized for the input signal, and presented as fold enrichment with respect to the IgG negative control. Data are expressed as the mean ± SD (n = 2).

    Article Snippet: The primary antibodies against SOX9 (# sc-20095), aggrecan (ACAN, # sc-33695), HIF-1α (H1α67, # sc-53546), NFATc1 (7A6, # sc-7294; H-10, # sc-1783; H-110, # sc-13033), lamin A/C (E-1, # sc-376248), and normal mouse IgG 1 (# sc-3877) were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, United States), while anti-collagen type II α1 chain (COL2a1; # ab3092) antibody was purchased from Abcam (Cambridge, United Kingdom), anti-HIF-1α (H1 alpha67, # NB100–134) was purchased from Novus Biologicals (Centennial, CO, United States), anti-P2X7R C-terminal (# APR-004) was purchased from Alomone Labs (Jerusalem, Israel), and anti-P2X7R C-terminal (#P8232) and anti-P2X7R extracellular loop (#P9122) were purchased from Sigma-Aldrich, Merck KGaA (Milan, Italy).

    Techniques: Expressing, Immunocytochemistry, Cell Culture, Software, Quantitative RT-PCR, Immunostaining, Binding Assay, Amplification, Negative Control

    P2X7R expression in blood of patients with GBS and healthy controls. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood samples, and P2X7R expression was examined using flow cytometry and qRT-PCR. (A) P2X7R mRNA levels were elevated in the PBMCs collected from patients with GBS compared to those in PBMCs collected from the healthy controls ( n = 8 per group). (B-D) P2X7R expression increased on CD4 + T cells in the PBMCs collected from patients with GBS, but no significant increase was noted on CD8 + T cells or monocytes (CD14 + ) compared to healthy controls ( n = 7 per group). HC, healthy control; GBS, Guillain–Barre Syndrome. * p < 0.05, ** p < 0.01

    Journal: Journal of Neuroinflammation

    Article Title: P2X7 receptor antagonists modulate experimental autoimmune neuritis via regulation of NLRP3 inflammasome activation and Th17 and Th1 cell differentiation

    doi: 10.1186/s12974-024-03057-z

    Figure Lengend Snippet: P2X7R expression in blood of patients with GBS and healthy controls. Human peripheral blood mononuclear cells (PBMCs) were isolated from blood samples, and P2X7R expression was examined using flow cytometry and qRT-PCR. (A) P2X7R mRNA levels were elevated in the PBMCs collected from patients with GBS compared to those in PBMCs collected from the healthy controls ( n = 8 per group). (B-D) P2X7R expression increased on CD4 + T cells in the PBMCs collected from patients with GBS, but no significant increase was noted on CD8 + T cells or monocytes (CD14 + ) compared to healthy controls ( n = 7 per group). HC, healthy control; GBS, Guillain–Barre Syndrome. * p < 0.05, ** p < 0.01

    Article Snippet: Non-specific binding sites were blocked with 3% BSA for 1 h. Next, the sections were incubated with the following antibodies overnight at 4 °C: rabbit anti-P2X7R (1:100, Alomone Labs), rabbit anti-IL-17 (1:200, Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-CD4 (1:50, BioLegend), mouse anti-CD8 (1:50, BioLegend), mouse anti-CD68 (1:50, BioLegend), anti-NLRP3 (1:200, Thermo Fisher) and mouse anti-SOX10 (1:100, Thermo Fisher Scientific).

    Techniques: Expressing, Isolation, Flow Cytometry, Quantitative RT-PCR

    P2X7R expression was significantly elevated in CD4 + T cells in EAN rats. P2X7R expression in T cells and macrophages in control (unimmunized group) and EAN rats at day 18. (A-B) Fluorescence photomicrographs showing P2X7R expression and quantification in CD4 + T cells, CD8 + T cells and macrophages (CD68 + ) in the sciatic nerves of EAN and control rats. Scale bars, 20 μm. (C) Graph showing the number of P2X7R-positive cells in sciatic nerve sections obtained from control and EAN rats. (D-E) Flow cytometry analysis of P2X7R expression and quantification on CD4 + T cells, CD8 + T cells and macrophages in the spleen of EAN and control rats. Experiments were performed in triplicate ( n = 6 per replicate) with similar results. * p < 0.05, ** p < 0.01

    Journal: Journal of Neuroinflammation

    Article Title: P2X7 receptor antagonists modulate experimental autoimmune neuritis via regulation of NLRP3 inflammasome activation and Th17 and Th1 cell differentiation

    doi: 10.1186/s12974-024-03057-z

    Figure Lengend Snippet: P2X7R expression was significantly elevated in CD4 + T cells in EAN rats. P2X7R expression in T cells and macrophages in control (unimmunized group) and EAN rats at day 18. (A-B) Fluorescence photomicrographs showing P2X7R expression and quantification in CD4 + T cells, CD8 + T cells and macrophages (CD68 + ) in the sciatic nerves of EAN and control rats. Scale bars, 20 μm. (C) Graph showing the number of P2X7R-positive cells in sciatic nerve sections obtained from control and EAN rats. (D-E) Flow cytometry analysis of P2X7R expression and quantification on CD4 + T cells, CD8 + T cells and macrophages in the spleen of EAN and control rats. Experiments were performed in triplicate ( n = 6 per replicate) with similar results. * p < 0.05, ** p < 0.01

    Article Snippet: Non-specific binding sites were blocked with 3% BSA for 1 h. Next, the sections were incubated with the following antibodies overnight at 4 °C: rabbit anti-P2X7R (1:100, Alomone Labs), rabbit anti-IL-17 (1:200, Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-CD4 (1:50, BioLegend), mouse anti-CD8 (1:50, BioLegend), mouse anti-CD68 (1:50, BioLegend), anti-NLRP3 (1:200, Thermo Fisher) and mouse anti-SOX10 (1:100, Thermo Fisher Scientific).

    Techniques: Expressing, Fluorescence, Flow Cytometry

    BBG treatment suppressed the P2X7R/NLRP3 pathway in CD4 + T cells in EAN. Proteins were obtained from sciatic nerves at the peak of EAN and subjected to western blot analysis and quantification. NLRP3 expression in immune cells was analyzed through flow cytometry and immunofluorescence. (A) Representative western blots of P2X7R, NLRP3, IL-1β and Caspase-1 protein expression in sciatic nerve. (B-E) Protein levels were quantified via densitometric analysis of immunoreactive bands in western blots using ImageJ software and were normalized to that of GAPDH. (F-G) Flow cytometry analysis and NLRP3 quantification on CD4 + T cells in splenic MNCs of control (unimmunized group), vehicle and BBG-treated rats. (H) Immunofluorescence photomicrographs of NLRP3 expression in CD4 + T cells in the sciatic nerves of control, vehicle, and BBG-treated EAN rats. (I) Number of NLRP3-positive cells in sciatic nerve sections obtained from control rats, vehicle and BBG-treated EAN rats. (J) NLRP3 quantification on CD4 + T cells. Scale bars, 20 μm. Experiments were performed in triplicate ( n = 6 per replicate) with similar results. Control group, unimmunized group; BBG-P, preventative BBG group; BBG-T, therapeutic BBG group. * p < 0.05, ** p < 0.01

    Journal: Journal of Neuroinflammation

    Article Title: P2X7 receptor antagonists modulate experimental autoimmune neuritis via regulation of NLRP3 inflammasome activation and Th17 and Th1 cell differentiation

    doi: 10.1186/s12974-024-03057-z

    Figure Lengend Snippet: BBG treatment suppressed the P2X7R/NLRP3 pathway in CD4 + T cells in EAN. Proteins were obtained from sciatic nerves at the peak of EAN and subjected to western blot analysis and quantification. NLRP3 expression in immune cells was analyzed through flow cytometry and immunofluorescence. (A) Representative western blots of P2X7R, NLRP3, IL-1β and Caspase-1 protein expression in sciatic nerve. (B-E) Protein levels were quantified via densitometric analysis of immunoreactive bands in western blots using ImageJ software and were normalized to that of GAPDH. (F-G) Flow cytometry analysis and NLRP3 quantification on CD4 + T cells in splenic MNCs of control (unimmunized group), vehicle and BBG-treated rats. (H) Immunofluorescence photomicrographs of NLRP3 expression in CD4 + T cells in the sciatic nerves of control, vehicle, and BBG-treated EAN rats. (I) Number of NLRP3-positive cells in sciatic nerve sections obtained from control rats, vehicle and BBG-treated EAN rats. (J) NLRP3 quantification on CD4 + T cells. Scale bars, 20 μm. Experiments were performed in triplicate ( n = 6 per replicate) with similar results. Control group, unimmunized group; BBG-P, preventative BBG group; BBG-T, therapeutic BBG group. * p < 0.05, ** p < 0.01

    Article Snippet: Non-specific binding sites were blocked with 3% BSA for 1 h. Next, the sections were incubated with the following antibodies overnight at 4 °C: rabbit anti-P2X7R (1:100, Alomone Labs), rabbit anti-IL-17 (1:200, Thermo Fisher Scientific, Waltham, MA, USA), mouse anti-CD4 (1:50, BioLegend), mouse anti-CD8 (1:50, BioLegend), mouse anti-CD68 (1:50, BioLegend), anti-NLRP3 (1:200, Thermo Fisher) and mouse anti-SOX10 (1:100, Thermo Fisher Scientific).

    Techniques: Western Blot, Expressing, Flow Cytometry, Immunofluorescence, Software