p2rx1  (Alomone Labs)


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    Alomone Labs p2rx1
    Repression of <t>P2rx1</t> by progressive vascular endothelial damage. ( a – c ) RQ-PCR ( a ) and western blot ( b , c ) analysis of the expressions of the cholinergic receptor muscarinic 2 ( Chrm2 ), cholinergic receptor muscarinic 3 ( Chrm3 ), and purinergic receptor P2X 1 ( P2rx1 ) genes in the bladders of rats in the indicated groups. Expression levels of the indicated transcripts are presented as % Gapdh . For western blot analysis, β-actin was used as a loading control, and the expression levels of the indicated proteins were normalized to the β-actin value. ( d , e ) Representative images ( d ) and quantification analysis ( e ) of the immunohistochemical staining for the P2rx1 protein (original magnification ×200, scale bar = 200 μm). All quantification results are presented as mean ± SEM. *p
    P2rx1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/p2rx1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    p2rx1 - by Bioz Stars, 2022-07
    93/100 stars

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    1) Product Images from "Induction of detrusor underactivity by extensive vascular endothelial damages of iliac arteries in a rat model and its pathophysiology in the genetic levels"

    Article Title: Induction of detrusor underactivity by extensive vascular endothelial damages of iliac arteries in a rat model and its pathophysiology in the genetic levels

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52811-4

    Repression of P2rx1 by progressive vascular endothelial damage. ( a – c ) RQ-PCR ( a ) and western blot ( b , c ) analysis of the expressions of the cholinergic receptor muscarinic 2 ( Chrm2 ), cholinergic receptor muscarinic 3 ( Chrm3 ), and purinergic receptor P2X 1 ( P2rx1 ) genes in the bladders of rats in the indicated groups. Expression levels of the indicated transcripts are presented as % Gapdh . For western blot analysis, β-actin was used as a loading control, and the expression levels of the indicated proteins were normalized to the β-actin value. ( d , e ) Representative images ( d ) and quantification analysis ( e ) of the immunohistochemical staining for the P2rx1 protein (original magnification ×200, scale bar = 200 μm). All quantification results are presented as mean ± SEM. *p
    Figure Legend Snippet: Repression of P2rx1 by progressive vascular endothelial damage. ( a – c ) RQ-PCR ( a ) and western blot ( b , c ) analysis of the expressions of the cholinergic receptor muscarinic 2 ( Chrm2 ), cholinergic receptor muscarinic 3 ( Chrm3 ), and purinergic receptor P2X 1 ( P2rx1 ) genes in the bladders of rats in the indicated groups. Expression levels of the indicated transcripts are presented as % Gapdh . For western blot analysis, β-actin was used as a loading control, and the expression levels of the indicated proteins were normalized to the β-actin value. ( d , e ) Representative images ( d ) and quantification analysis ( e ) of the immunohistochemical staining for the P2rx1 protein (original magnification ×200, scale bar = 200 μm). All quantification results are presented as mean ± SEM. *p

    Techniques Used: Polymerase Chain Reaction, Western Blot, Expressing, Immunohistochemistry, Staining

    2) Product Images from "Enhanced electrical field stimulated nitrergic and purinergic vasoreactivity in distal versus proximal internal pudendal arteries"

    Article Title: Enhanced electrical field stimulated nitrergic and purinergic vasoreactivity in distal versus proximal internal pudendal arteries

    Journal: The journal of sexual medicine

    doi: 10.1016/j.jsxm.2017.09.013

    Effectiveness of P2X1 and P2Y1 antagonists (A) NF449 (10 −5 M) eliminated α,ß-MetATP induced contractions in proximal (n=6) and (B) distal IPA (n=6). (C) MRS2500 (10 −6 M) did not impair ADP induced relaxation in proximal IPA (n=6) but (D) did decrease relaxation in distal IPA (n=6). (E) MRS2500 did not impair 2-MeSADP induced proximal IPA relaxation (n=8) or (F) reduce distal IPA relaxation to 2-MeSADP (n=8). For all values, mean ± SEM and *p
    Figure Legend Snippet: Effectiveness of P2X1 and P2Y1 antagonists (A) NF449 (10 −5 M) eliminated α,ß-MetATP induced contractions in proximal (n=6) and (B) distal IPA (n=6). (C) MRS2500 (10 −6 M) did not impair ADP induced relaxation in proximal IPA (n=6) but (D) did decrease relaxation in distal IPA (n=6). (E) MRS2500 did not impair 2-MeSADP induced proximal IPA relaxation (n=8) or (F) reduce distal IPA relaxation to 2-MeSADP (n=8). For all values, mean ± SEM and *p

    Techniques Used: Indirect Immunoperoxidase Assay

    No differences in P2X1 and P2Y1 receptor protein expression (A) P2X1 receptor expression in proximal and distal IPA normalized to ß-actin. (B) P2Y1 receptor expression in proximal and distal IPA normalized to ß-actin. For all values, n=8/group and mean ± SEM.
    Figure Legend Snippet: No differences in P2X1 and P2Y1 receptor protein expression (A) P2X1 receptor expression in proximal and distal IPA normalized to ß-actin. (B) P2Y1 receptor expression in proximal and distal IPA normalized to ß-actin. For all values, n=8/group and mean ± SEM.

    Techniques Used: Expressing, Indirect Immunoperoxidase Assay

    Purinergic antagonism improves distal IPA NANC relaxation (A) NANC relaxation with addition of selective P2X1 antagonist, NF449, in proximal and (B) distal IPA (n=8/segment). (C) NANC relaxation with addition of selective P2Y1 antagonist, MRS2500, in proximal and (D) distal IPA (n=8/segment). (E) Combined P2X1 and P2Y1 receptor antagonism in NANC relaxation using NF449 and MRS2500 in proximal and (F) distal IPA (n=8/segment). All values are represented as percent decrease from PE precontraction. For all values, mean ± SEM and *p
    Figure Legend Snippet: Purinergic antagonism improves distal IPA NANC relaxation (A) NANC relaxation with addition of selective P2X1 antagonist, NF449, in proximal and (B) distal IPA (n=8/segment). (C) NANC relaxation with addition of selective P2Y1 antagonist, MRS2500, in proximal and (D) distal IPA (n=8/segment). (E) Combined P2X1 and P2Y1 receptor antagonism in NANC relaxation using NF449 and MRS2500 in proximal and (F) distal IPA (n=8/segment). All values are represented as percent decrease from PE precontraction. For all values, mean ± SEM and *p

    Techniques Used: Indirect Immunoperoxidase Assay

    3) Product Images from "Induction of detrusor underactivity by extensive vascular endothelial damages of iliac arteries in a rat model and its pathophysiology in the genetic levels"

    Article Title: Induction of detrusor underactivity by extensive vascular endothelial damages of iliac arteries in a rat model and its pathophysiology in the genetic levels

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-52811-4

    Repression of P2rx1 by progressive vascular endothelial damage. ( a – c ) RQ-PCR ( a ) and western blot ( b , c ) analysis of the expressions of the cholinergic receptor muscarinic 2 ( Chrm2 ), cholinergic receptor muscarinic 3 ( Chrm3 ), and purinergic receptor P2X 1 ( P2rx1 ) genes in the bladders of rats in the indicated groups. Expression levels of the indicated transcripts are presented as % Gapdh . For western blot analysis, β-actin was used as a loading control, and the expression levels of the indicated proteins were normalized to the β-actin value. ( d , e ) Representative images ( d ) and quantification analysis ( e ) of the immunohistochemical staining for the P2rx1 protein (original magnification ×200, scale bar = 200 μm). All quantification results are presented as mean ± SEM. *p
    Figure Legend Snippet: Repression of P2rx1 by progressive vascular endothelial damage. ( a – c ) RQ-PCR ( a ) and western blot ( b , c ) analysis of the expressions of the cholinergic receptor muscarinic 2 ( Chrm2 ), cholinergic receptor muscarinic 3 ( Chrm3 ), and purinergic receptor P2X 1 ( P2rx1 ) genes in the bladders of rats in the indicated groups. Expression levels of the indicated transcripts are presented as % Gapdh . For western blot analysis, β-actin was used as a loading control, and the expression levels of the indicated proteins were normalized to the β-actin value. ( d , e ) Representative images ( d ) and quantification analysis ( e ) of the immunohistochemical staining for the P2rx1 protein (original magnification ×200, scale bar = 200 μm). All quantification results are presented as mean ± SEM. *p

    Techniques Used: Polymerase Chain Reaction, Western Blot, Expressing, Immunohistochemistry, Staining

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    Alomone Labs anti p2x1
    Single-cell RNA sequencing reveals heterogeneity and distinct subsets of fibroblasts. a Diagram depicting the generation of tdTomato rPβC mouse and PDGFRβ + cell-specific expression of tdTomato from 8-week old and their analyses at 10-week old. b Illustration of droplet-based single-cell RNA sequencing (scRNA-seq) of PDGFRβ + tdTomato + IntSCs sorted from the small intestine of tdTomato rPβC mice. c Visualization of unsupervised clustering of 7 distinct PDGFRβ + IntSC clusters by Uniform manifold approximation and projection (UMAP) in the small intestine of tdTomato rPβC mice. vFB, intestinal villi fibroblast; FB, fibroblast; SMC, smooth muscle cell; MC, mural cell. d Heatmap displaying the scaled expression patterns of top ten differentially expressed genes for random sampled cells (maximum thousand cells) for each indicated clusters. e List of representative marker genes of each of the seven PDGFRβ + IntSC clusters (left) and violin plots (right) showing the expression of top-ranking marker genes for each cluster. Log normalized read counts as y -axis (normalized expression). f UMAP visualization of unsupervised clustering of seven distinct PDGFRβ + IntSC clusters in the small intestine of Lats1/2 iΔ - tdTomato rPβC mice. g Gene expression levels of Vegfc and Vegfa in tdTomato rPβC and Lats1/2 iΔ - tdTomato rPβC mice projected on UMAP plot. Note that specific subsets of PDGFRβ + IntSCs (vFB1-3) in the small intestine of Lats1/2 iΔ - tdTomato rPβC mice show higher expression of Vegfc compared with those of tdTomato rPβC mice. h , i Representative images of PDGFRβ + IntSCs in WT mouse reveal expressions of each fibroblast-specific markers: PAI-1 + vFB1, Serpina3n + vFB2, <t>P2X1</t> + vFB3, Ackr4 + or Grem1 + FB4, and PDGFRα + Sox6 + FB5. Each white box in the lower left corner is a magnified view. White asterisks indicate lacteals and white arrowheads indicate each fibroblast cluster-specific cell type stained with the indicated marker. Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 20 μm. j Schematic images depicting the anatomic distribution of indicated markers for vFB1-3, FB4, FB5, SMC, MC, capillary plexus, and lacteal in intestinal villi of adult WT mouse. vFB1-3 are uniformly distributed around the lacteal, whereas FB4 is mainly located in the submucosal area and FB5 is mostly placed under the intestinal epithelium. Black dashed boxes are magnified in the right panels.
    Anti P2x1, supplied by Alomone Labs, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti p2x1/product/Alomone Labs
    Average 93 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
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    Single-cell RNA sequencing reveals heterogeneity and distinct subsets of fibroblasts. a Diagram depicting the generation of tdTomato rPβC mouse and PDGFRβ + cell-specific expression of tdTomato from 8-week old and their analyses at 10-week old. b Illustration of droplet-based single-cell RNA sequencing (scRNA-seq) of PDGFRβ + tdTomato + IntSCs sorted from the small intestine of tdTomato rPβC mice. c Visualization of unsupervised clustering of 7 distinct PDGFRβ + IntSC clusters by Uniform manifold approximation and projection (UMAP) in the small intestine of tdTomato rPβC mice. vFB, intestinal villi fibroblast; FB, fibroblast; SMC, smooth muscle cell; MC, mural cell. d Heatmap displaying the scaled expression patterns of top ten differentially expressed genes for random sampled cells (maximum thousand cells) for each indicated clusters. e List of representative marker genes of each of the seven PDGFRβ + IntSC clusters (left) and violin plots (right) showing the expression of top-ranking marker genes for each cluster. Log normalized read counts as y -axis (normalized expression). f UMAP visualization of unsupervised clustering of seven distinct PDGFRβ + IntSC clusters in the small intestine of Lats1/2 iΔ - tdTomato rPβC mice. g Gene expression levels of Vegfc and Vegfa in tdTomato rPβC and Lats1/2 iΔ - tdTomato rPβC mice projected on UMAP plot. Note that specific subsets of PDGFRβ + IntSCs (vFB1-3) in the small intestine of Lats1/2 iΔ - tdTomato rPβC mice show higher expression of Vegfc compared with those of tdTomato rPβC mice. h , i Representative images of PDGFRβ + IntSCs in WT mouse reveal expressions of each fibroblast-specific markers: PAI-1 + vFB1, Serpina3n + vFB2, P2X1 + vFB3, Ackr4 + or Grem1 + FB4, and PDGFRα + Sox6 + FB5. Each white box in the lower left corner is a magnified view. White asterisks indicate lacteals and white arrowheads indicate each fibroblast cluster-specific cell type stained with the indicated marker. Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 20 μm. j Schematic images depicting the anatomic distribution of indicated markers for vFB1-3, FB4, FB5, SMC, MC, capillary plexus, and lacteal in intestinal villi of adult WT mouse. vFB1-3 are uniformly distributed around the lacteal, whereas FB4 is mainly located in the submucosal area and FB5 is mostly placed under the intestinal epithelium. Black dashed boxes are magnified in the right panels.

    Journal: Nature Communications

    Article Title: Distinct fibroblast subsets regulate lacteal integrity through YAP/TAZ-induced VEGF-C in intestinal villi

    doi: 10.1038/s41467-020-17886-y

    Figure Lengend Snippet: Single-cell RNA sequencing reveals heterogeneity and distinct subsets of fibroblasts. a Diagram depicting the generation of tdTomato rPβC mouse and PDGFRβ + cell-specific expression of tdTomato from 8-week old and their analyses at 10-week old. b Illustration of droplet-based single-cell RNA sequencing (scRNA-seq) of PDGFRβ + tdTomato + IntSCs sorted from the small intestine of tdTomato rPβC mice. c Visualization of unsupervised clustering of 7 distinct PDGFRβ + IntSC clusters by Uniform manifold approximation and projection (UMAP) in the small intestine of tdTomato rPβC mice. vFB, intestinal villi fibroblast; FB, fibroblast; SMC, smooth muscle cell; MC, mural cell. d Heatmap displaying the scaled expression patterns of top ten differentially expressed genes for random sampled cells (maximum thousand cells) for each indicated clusters. e List of representative marker genes of each of the seven PDGFRβ + IntSC clusters (left) and violin plots (right) showing the expression of top-ranking marker genes for each cluster. Log normalized read counts as y -axis (normalized expression). f UMAP visualization of unsupervised clustering of seven distinct PDGFRβ + IntSC clusters in the small intestine of Lats1/2 iΔ - tdTomato rPβC mice. g Gene expression levels of Vegfc and Vegfa in tdTomato rPβC and Lats1/2 iΔ - tdTomato rPβC mice projected on UMAP plot. Note that specific subsets of PDGFRβ + IntSCs (vFB1-3) in the small intestine of Lats1/2 iΔ - tdTomato rPβC mice show higher expression of Vegfc compared with those of tdTomato rPβC mice. h , i Representative images of PDGFRβ + IntSCs in WT mouse reveal expressions of each fibroblast-specific markers: PAI-1 + vFB1, Serpina3n + vFB2, P2X1 + vFB3, Ackr4 + or Grem1 + FB4, and PDGFRα + Sox6 + FB5. Each white box in the lower left corner is a magnified view. White asterisks indicate lacteals and white arrowheads indicate each fibroblast cluster-specific cell type stained with the indicated marker. Similar findings were observed in n = 4 mice from two independent experiments. Scale bars, 20 μm. j Schematic images depicting the anatomic distribution of indicated markers for vFB1-3, FB4, FB5, SMC, MC, capillary plexus, and lacteal in intestinal villi of adult WT mouse. vFB1-3 are uniformly distributed around the lacteal, whereas FB4 is mainly located in the submucosal area and FB5 is mostly placed under the intestinal epithelium. Black dashed boxes are magnified in the right panels.

    Article Snippet: The following primary and secondary antibodies were used in the immunostaining: anti-LYVE-1 (rabbit polyclonal, 11-034, Angiobio, 1:400); anti-CD31 (rat monoclonal, 557355, BD Biosciences, 1:400); anti-CD31 (hamster monoclonal, MAB1398Z, Merck, 1:400); anti-E-cadherin (goat polyclonal, AF748, R & D, 1:200); anti-Prox1 (goat polyclonal, AF2727, R & D, 1:400); anti-Prox1 (rabbit polyclonal, 102-PA32AG, ReliaTech, 1:400); anti-VE-cadherin (goat polyclonal, AF1002, R & D, 1:200); anti-PDGFRβ (rat monoclonal, ab91066, Abcam, 1:200); anti-PAI-1 (mouse monoclonal, sc-5297, Santa Cruz, 1:100); anti-Serpina3n (Goat polyclonal, AF4709, R & D, 1:200); anti-Fosb (rabbit monoclonal, 2251, Cell Signaling Technology, 1:400); anti-Shisa3 (rabbit polyclonal, TA320118, Origene, 1:400); anti-P2X1 (rabbit polyclonal, APR-001, Alomone labs, 1:800); anti-Ackr4 (rabbit polyclonal, SAB4502137, Sigma-Aldrich, 1:200); anti-Grem1 (goat polyclonal, AF956, R & D, 1:200); anti-Sox6 (rabbit polyclonal, ab30455, Abcam, 1:400); anti-PDGFRα (goat polyclonal, AF1062, R & D, 1:200); anti-YAP (rabbit monoclonal, 14074, Cell signaling, 1:200); anti-TAZ (rabbit polyclonal, HPA007415, Sigma-Aldrich, 1:200); anti-αSMA, Fluorescein Isothiocyanate (FITC)-conjugated (mouse monoclonal, F3777, Sigma-Aldrich, 1:1000); anti-VEGFR3 (goat polyclonal, AF743, R & D, 1:200); anti-VEGFR2 (goat polyclonal, AF644, R & D, 1:200); anti-PGP9.5 (rabbit monoclonal, 13179, Cell signaling, 1:400); anti-F4/80, FITC-conjugated (rat monoclonal, 1231007, Biolegend, 1:200); anti-CD3 (hamster monoclonal, 553058, BD Biosciences, 1:1000); anti-Desmin (rabbit polyclonal, AB907, Millipore, 1:400); and Alexa Fluor 488-, Alexa Fluor 594-, Alexa Fluor 647-conjugated anti-rabbit, anti-rat, anti-goat, anti-hamster secondary antibodies (diluted at a ratio of 1:1000) were purchased from Jackson ImmunoResearch.

    Techniques: RNA Sequencing Assay, Expressing, Mouse Assay, Marker, Staining

    Localization of 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modification of the human P2X1 receptor. (a) P2X1 homology model (based on zP2X4 crystal structure model – Kawate et al . 2009 ) depicted as a trimeric cartoon. Lysine residues modified DTSSP and identified by mass spectrometry are indicated in red. Lysine residue K70 that was not observed in any mass spectrometry runs is shown in dark grey. Residues observed and not modified by DTSSP in some runs are shown in grey. Residue K68 that was not observed to be modified by DTSSP (11/11) is shown in black. The serine residues 130, 286 (blue spheres) and tyrosine 274 (green spheres) were also modified by DTSSP. (b) Human P2X1 protein sequence with DTSSP modification of the P2X1 receptor marked as observed from mass spectrometry data. Transmembrane regions 1 and 2 are highlighted with a bold line. Residues are coloured as indicated in panel (a). Arrows indicate residues modified by DTSSP, an arrow with a cross show a residue that was not modified by DTSSP, and arrows with a question mark where it remains unknown either because of non-coverage of the protein sequence or modification not detected. Note that all the DTSSP modifications occur in the extracellular region.

    Journal: Journal of Neurochemistry

    Article Title: Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

    doi: 10.1111/jnc.12012

    Figure Lengend Snippet: Localization of 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modification of the human P2X1 receptor. (a) P2X1 homology model (based on zP2X4 crystal structure model – Kawate et al . 2009 ) depicted as a trimeric cartoon. Lysine residues modified DTSSP and identified by mass spectrometry are indicated in red. Lysine residue K70 that was not observed in any mass spectrometry runs is shown in dark grey. Residues observed and not modified by DTSSP in some runs are shown in grey. Residue K68 that was not observed to be modified by DTSSP (11/11) is shown in black. The serine residues 130, 286 (blue spheres) and tyrosine 274 (green spheres) were also modified by DTSSP. (b) Human P2X1 protein sequence with DTSSP modification of the P2X1 receptor marked as observed from mass spectrometry data. Transmembrane regions 1 and 2 are highlighted with a bold line. Residues are coloured as indicated in panel (a). Arrows indicate residues modified by DTSSP, an arrow with a cross show a residue that was not modified by DTSSP, and arrows with a question mark where it remains unknown either because of non-coverage of the protein sequence or modification not detected. Note that all the DTSSP modifications occur in the extracellular region.

    Article Snippet: Proteins were transferred to nitrocellulose, probed with P2X1 primary antibody (1 : 1000; Alomone Labs) followed by secondary goat anti-rabbit antibody (A6154, 1 : 3500 dilution; Sigma).

    Techniques: Modification, Mass Spectrometry, Sequencing

    Effect of 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modification on Human P2X1 receptor function. (a) Application of 100 μM ATP to Xenopus oocytes expressing P2X1 wildtype receptors evoked a large inward current recorded by two electrode voltage clamp. Pre-incubation with 100 μM DTSSP for 30 min almost abolished responses. Inset traces are representative of normalized responses to 100 μM ATP in the presence or absence of 100 μM DTSSP indicating no significant change in the current time course. (b) Pooled electrophysiology data depicting the decrease in channel function post-DTSSP treatment. (c) P2X1 receptor ATP-binding site analysis utilizing uv cross-linked 32 P 2Azido ATP (2AzATP) shows a marked reduction in radioactivity of the P2X1 receptor protein band following pre-treatment with 100 μM DTSSP. (d) Pooled densitometry data collected from autoradiography of the DTSSP treated and non-treated 2AzATP radioactive P2X1 receptor bands ( n = 4) *** p

    Journal: Journal of Neurochemistry

    Article Title: Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

    doi: 10.1111/jnc.12012

    Figure Lengend Snippet: Effect of 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modification on Human P2X1 receptor function. (a) Application of 100 μM ATP to Xenopus oocytes expressing P2X1 wildtype receptors evoked a large inward current recorded by two electrode voltage clamp. Pre-incubation with 100 μM DTSSP for 30 min almost abolished responses. Inset traces are representative of normalized responses to 100 μM ATP in the presence or absence of 100 μM DTSSP indicating no significant change in the current time course. (b) Pooled electrophysiology data depicting the decrease in channel function post-DTSSP treatment. (c) P2X1 receptor ATP-binding site analysis utilizing uv cross-linked 32 P 2Azido ATP (2AzATP) shows a marked reduction in radioactivity of the P2X1 receptor protein band following pre-treatment with 100 μM DTSSP. (d) Pooled densitometry data collected from autoradiography of the DTSSP treated and non-treated 2AzATP radioactive P2X1 receptor bands ( n = 4) *** p

    Article Snippet: Proteins were transferred to nitrocellulose, probed with P2X1 primary antibody (1 : 1000; Alomone Labs) followed by secondary goat anti-rabbit antibody (A6154, 1 : 3500 dilution; Sigma).

    Techniques: Modification, Expressing, Incubation, Binding Assay, Radioactivity, Autoradiography

    Purification and mass spectrometry analysis of the Human P2X1 receptor. (a) Anti-P2X1 receptor antibody western blot analysis of 3X FLAG peptide eluted fractions from anti-FLAG agarose beads. FT, flow through; W1–2, washes; E1–E10, FLAG peptide eluted fractions; E11–13, 0.1 M Glycine pH 3.5 eluted fractions. P2X1 protein of the correct mass is observed in fractions E2–E8. (b) InstantBlue (Expedeon) stained gel of eluted fractions from anti-FLAG agarose beads (lanes labelled as Fig. 1a ). (c) Combined and concentrated fractions were run on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with InstantBlue (Expedeon). Clean purified FLAG tagged P2X1 protein can be observed along with PNGase F deglycosylated P2X1 receptor protein and the PNGase F protein (star indicates confirmed by mass spectrometry) (d) Identification of the P2X1 receptor with 26.8 ± 6% percentage coverage on single mass spectrometry runs was achieved with trypsin digest though other enzymes were tested (e.g. chymotrypsin, 11.2 ± 5% coverage, data not shown). Coverage of the P2X1 receptor protein was increased (26.8 ± 6% vs. 37.8 ± 3%) by deglycosylating the receptors with PNGase F. Use of Orbitrap versus Qtrap mass spectrometer increased mass accuracy and peptide identification increasing coverage even further on average to 59.2 ± 2%. (e) Human P2X1 receptor protein sequence showing the total coverage of all observed peptides (26 runs). Transmembrane regions 1 and 2 are highlighted with a bold line. Amino acid residues shown in bold lowercase were not observed on mass spectrometry of the P2X1 receptor protein most probably because their masses were below the limit of detection (∼500 Da) (Table S1). Other areas of predicted low mass were only observed as a result of partial digestion and therefore identified as part of a larger peptide mass. Vertical lines indicate sites for trypsin digestion at arginine and lysine residues.

    Journal: Journal of Neurochemistry

    Article Title: Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

    doi: 10.1111/jnc.12012

    Figure Lengend Snippet: Purification and mass spectrometry analysis of the Human P2X1 receptor. (a) Anti-P2X1 receptor antibody western blot analysis of 3X FLAG peptide eluted fractions from anti-FLAG agarose beads. FT, flow through; W1–2, washes; E1–E10, FLAG peptide eluted fractions; E11–13, 0.1 M Glycine pH 3.5 eluted fractions. P2X1 protein of the correct mass is observed in fractions E2–E8. (b) InstantBlue (Expedeon) stained gel of eluted fractions from anti-FLAG agarose beads (lanes labelled as Fig. 1a ). (c) Combined and concentrated fractions were run on a 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and stained with InstantBlue (Expedeon). Clean purified FLAG tagged P2X1 protein can be observed along with PNGase F deglycosylated P2X1 receptor protein and the PNGase F protein (star indicates confirmed by mass spectrometry) (d) Identification of the P2X1 receptor with 26.8 ± 6% percentage coverage on single mass spectrometry runs was achieved with trypsin digest though other enzymes were tested (e.g. chymotrypsin, 11.2 ± 5% coverage, data not shown). Coverage of the P2X1 receptor protein was increased (26.8 ± 6% vs. 37.8 ± 3%) by deglycosylating the receptors with PNGase F. Use of Orbitrap versus Qtrap mass spectrometer increased mass accuracy and peptide identification increasing coverage even further on average to 59.2 ± 2%. (e) Human P2X1 receptor protein sequence showing the total coverage of all observed peptides (26 runs). Transmembrane regions 1 and 2 are highlighted with a bold line. Amino acid residues shown in bold lowercase were not observed on mass spectrometry of the P2X1 receptor protein most probably because their masses were below the limit of detection (∼500 Da) (Table S1). Other areas of predicted low mass were only observed as a result of partial digestion and therefore identified as part of a larger peptide mass. Vertical lines indicate sites for trypsin digestion at arginine and lysine residues.

    Article Snippet: Proteins were transferred to nitrocellulose, probed with P2X1 primary antibody (1 : 1000; Alomone Labs) followed by secondary goat anti-rabbit antibody (A6154, 1 : 3500 dilution; Sigma).

    Techniques: Purification, Mass Spectrometry, Western Blot, Staining, Polyacrylamide Gel Electrophoresis, SDS Page, Sequencing

    Site directed mutagenesis of human P2X1 receptor to discover the molecular basis for 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) inhibition. (a) Cartoon representation of P2X1 receptor structure highlighting the residues K199 and K221 (blue spheres) positioned on different subunits approximately 12 angstroms apart. The diameter of the circle shown is approximately 24 angstroms. Residues in yellow correspond to positions 196 and 320 in adjacent subunits where introduced cysteine residues form a disulphide bond and inhibit channel activation. Red spheres correspond to lysine residues that when mutated had no effect on DTSSP inhibition of ATP evoked responses. (b) Combining mass spectrometry and crystal structure to map possible cross-linked pairs, multiple mutations were designed to discover the residues responsible for DTSSP inhibition and cross-linking. Residues 70 : 140; 70 : 309; 70 : 286; 140 : 215; 190 : 283; 190 : 286; 199 : 221 and 322 : 322 are all within 12 angstroms and on separate P2X1 subunits and therefore possible candidates for causing functional inhibition and dimer/trimer formation with DTSSP. Only double mutant K199R:K221R showed a significant reduction of DTSSP inhibition also reflected in the single mutants K199R and K221R ( n = 3–25). No mutations were observed to disrupt the DTSSP formation of dimers/trimers on western blot (data not shown). Inset example trace data for human P2X1 wildtype and P2X1 double mutant K199R: K221R in the presence and absence of 100 μM DTSSP ** p

    Journal: Journal of Neurochemistry

    Article Title: Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

    doi: 10.1111/jnc.12012

    Figure Lengend Snippet: Site directed mutagenesis of human P2X1 receptor to discover the molecular basis for 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) inhibition. (a) Cartoon representation of P2X1 receptor structure highlighting the residues K199 and K221 (blue spheres) positioned on different subunits approximately 12 angstroms apart. The diameter of the circle shown is approximately 24 angstroms. Residues in yellow correspond to positions 196 and 320 in adjacent subunits where introduced cysteine residues form a disulphide bond and inhibit channel activation. Red spheres correspond to lysine residues that when mutated had no effect on DTSSP inhibition of ATP evoked responses. (b) Combining mass spectrometry and crystal structure to map possible cross-linked pairs, multiple mutations were designed to discover the residues responsible for DTSSP inhibition and cross-linking. Residues 70 : 140; 70 : 309; 70 : 286; 140 : 215; 190 : 283; 190 : 286; 199 : 221 and 322 : 322 are all within 12 angstroms and on separate P2X1 subunits and therefore possible candidates for causing functional inhibition and dimer/trimer formation with DTSSP. Only double mutant K199R:K221R showed a significant reduction of DTSSP inhibition also reflected in the single mutants K199R and K221R ( n = 3–25). No mutations were observed to disrupt the DTSSP formation of dimers/trimers on western blot (data not shown). Inset example trace data for human P2X1 wildtype and P2X1 double mutant K199R: K221R in the presence and absence of 100 μM DTSSP ** p

    Article Snippet: Proteins were transferred to nitrocellulose, probed with P2X1 primary antibody (1 : 1000; Alomone Labs) followed by secondary goat anti-rabbit antibody (A6154, 1 : 3500 dilution; Sigma).

    Techniques: Mutagenesis, Inhibition, Activation Assay, Mass Spectrometry, Functional Assay, Western Blot

    Phosphorylation status of the Human P2X1 receptor. (a) P2X1 receptor protein sequence of the N- and C-termini. Intracellular modifications made by membrane permeable Dithio bis (sulfosuccinimidylpropionate) (DSP) were identified by mass spectrometry and are indicated on the protein sequence. Membrane impermeable 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modifications were only observed on extracellular P2X1 protein residues. DSP modification highlights the lipid/transmembrane boundaries with K27 and K28 modified showing accessibility. In silico analysis of the N-terminal protein sequence reveals residues Y16 and T18 as potential phosphorylation sites (bold type). Mass spectrometry analysis did not show modification at these residues even after enriching for phosphorylated peptides. In silico analysis of the C-terminal protein sequence reveals tyrosine residues Y362, 363 and 370 and serine and threonine residues 386–389 and 398–399 as potential phosphorylation sites (bold type). Phosphorylation was initially observed at residues S387 and S388 and residues S388, T389 and S399 were identified on mass spectrometry runs enriched for phosphorylated peptides (P *). These residues were the only phosphorylated residues observed on mass spectrometry of the P2X1 receptor protein. (b) ATP evoked currents (period indicated by bar) from P2X1 wildtype and mutant receptors (S387A and S388A – SS-AA, S387A, S388A and T389A – SST-AAA). Traces show reproducible response evoked at a 5-minute interval and level of recovery from desensitization at 60 s between ATP applications. (c, d) Time to peak (10–90%) and decay time (100–50%) of currents evoked by 100 μM ATP for wildtype and mutant P2X1 receptors. (e) Recovery from desensitization at 60 s for wildtype and mutant P2X1 receptors. * p

    Journal: Journal of Neurochemistry

    Article Title: Mass spectrometry analysis of human P2X1 receptors; insight into phosphorylation, modelling and conformational changes

    doi: 10.1111/jnc.12012

    Figure Lengend Snippet: Phosphorylation status of the Human P2X1 receptor. (a) P2X1 receptor protein sequence of the N- and C-termini. Intracellular modifications made by membrane permeable Dithio bis (sulfosuccinimidylpropionate) (DSP) were identified by mass spectrometry and are indicated on the protein sequence. Membrane impermeable 3,3′-Dithio bis (sulfosuccinimidylpropionate) (DTSSP) modifications were only observed on extracellular P2X1 protein residues. DSP modification highlights the lipid/transmembrane boundaries with K27 and K28 modified showing accessibility. In silico analysis of the N-terminal protein sequence reveals residues Y16 and T18 as potential phosphorylation sites (bold type). Mass spectrometry analysis did not show modification at these residues even after enriching for phosphorylated peptides. In silico analysis of the C-terminal protein sequence reveals tyrosine residues Y362, 363 and 370 and serine and threonine residues 386–389 and 398–399 as potential phosphorylation sites (bold type). Phosphorylation was initially observed at residues S387 and S388 and residues S388, T389 and S399 were identified on mass spectrometry runs enriched for phosphorylated peptides (P *). These residues were the only phosphorylated residues observed on mass spectrometry of the P2X1 receptor protein. (b) ATP evoked currents (period indicated by bar) from P2X1 wildtype and mutant receptors (S387A and S388A – SS-AA, S387A, S388A and T389A – SST-AAA). Traces show reproducible response evoked at a 5-minute interval and level of recovery from desensitization at 60 s between ATP applications. (c, d) Time to peak (10–90%) and decay time (100–50%) of currents evoked by 100 μM ATP for wildtype and mutant P2X1 receptors. (e) Recovery from desensitization at 60 s for wildtype and mutant P2X1 receptors. * p

    Article Snippet: Proteins were transferred to nitrocellulose, probed with P2X1 primary antibody (1 : 1000; Alomone Labs) followed by secondary goat anti-rabbit antibody (A6154, 1 : 3500 dilution; Sigma).

    Techniques: Sequencing, Mass Spectrometry, Modification, In Silico, Mutagenesis

    Double labeling of P2X1 receptor-ir and NeuN-ir in the submucosal plexus of the jejunum, ileum, and distal colon of the mouse. a , c , e P2X1 receptor-ir neurons (red) in the jejunum, ileum, and distal colon, respectively. b , d , f The merged images of a , c , and e and NeuN-ir neurons (green) in the same regions of a , c , and e , respectively. An arrow indicates a double-labeled neuron. All the scale bars = 60 μm

    Journal: Purinergic Signalling

    Article Title: Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human

    doi: 10.1007/s11302-018-9615-6

    Figure Lengend Snippet: Double labeling of P2X1 receptor-ir and NeuN-ir in the submucosal plexus of the jejunum, ileum, and distal colon of the mouse. a , c , e P2X1 receptor-ir neurons (red) in the jejunum, ileum, and distal colon, respectively. b , d , f The merged images of a , c , and e and NeuN-ir neurons (green) in the same regions of a , c , and e , respectively. An arrow indicates a double-labeled neuron. All the scale bars = 60 μm

    Article Snippet: The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2) for 30 min, followed by incubation with different combinations of P2X1 antibody (Alomone, Israel, rabbit anti-rat) diluted 1:200, SPMA antibody (mouse anti-rat; Santa Cruz) diluted 1:200, ICAM1 antibody (mouse anti-rat; Proteintech) diluted 1:100, NeuN antibody (mouse anti-rat; Chemicon) diluted 1:200, SOM antibody (mouse anti-rat; Abgent) diluted 1:200 in antiserum solution 2 (1% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2), at room temperature.

    Techniques: Labeling

    Expression of P2X1 receptors and SPMA in the mouse distal colon. a P2X1 receptor-ir. b SPMA-ir in the same region of a . c DAPI staining in the same region of a . d The merged image of a – c . e A high magnification of the region indicated by an arrow in d , a white arrow indicates a nerve fiber with P2X1 receptor-ir. f The double-labeled image of P2X1 receptor-ir and SPMA-ir in the same region of e , an arrow indicates a double-labeled blood vessel. The scale bars in a – d = 120 μm, in e , f = 60 μm

    Journal: Purinergic Signalling

    Article Title: Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human

    doi: 10.1007/s11302-018-9615-6

    Figure Lengend Snippet: Expression of P2X1 receptors and SPMA in the mouse distal colon. a P2X1 receptor-ir. b SPMA-ir in the same region of a . c DAPI staining in the same region of a . d The merged image of a – c . e A high magnification of the region indicated by an arrow in d , a white arrow indicates a nerve fiber with P2X1 receptor-ir. f The double-labeled image of P2X1 receptor-ir and SPMA-ir in the same region of e , an arrow indicates a double-labeled blood vessel. The scale bars in a – d = 120 μm, in e , f = 60 μm

    Article Snippet: The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2) for 30 min, followed by incubation with different combinations of P2X1 antibody (Alomone, Israel, rabbit anti-rat) diluted 1:200, SPMA antibody (mouse anti-rat; Santa Cruz) diluted 1:200, ICAM1 antibody (mouse anti-rat; Proteintech) diluted 1:100, NeuN antibody (mouse anti-rat; Chemicon) diluted 1:200, SOM antibody (mouse anti-rat; Abgent) diluted 1:200 in antiserum solution 2 (1% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2), at room temperature.

    Techniques: Expressing, Staining, Labeling

    Double labeling of P2X1 receptor-ir and NeuN-ir in the myenteric plexus of the stomach corpus, jejunum, ileum, and distal colon of the mouse. a P2X1 receptor-ir in the myenteric plexus of the stomach. b NeuN-ir in the same region of a . c The merge image of a and b . d – f The merged images of P2X1 receptor-ir and NeuN-ir double labeling in the jejunum, ileum, and distal colon. An arrow indicates a double-labeled neuron. All the scale bars = 60 μm

    Journal: Purinergic Signalling

    Article Title: Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human

    doi: 10.1007/s11302-018-9615-6

    Figure Lengend Snippet: Double labeling of P2X1 receptor-ir and NeuN-ir in the myenteric plexus of the stomach corpus, jejunum, ileum, and distal colon of the mouse. a P2X1 receptor-ir in the myenteric plexus of the stomach. b NeuN-ir in the same region of a . c The merge image of a and b . d – f The merged images of P2X1 receptor-ir and NeuN-ir double labeling in the jejunum, ileum, and distal colon. An arrow indicates a double-labeled neuron. All the scale bars = 60 μm

    Article Snippet: The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2) for 30 min, followed by incubation with different combinations of P2X1 antibody (Alomone, Israel, rabbit anti-rat) diluted 1:200, SPMA antibody (mouse anti-rat; Santa Cruz) diluted 1:200, ICAM1 antibody (mouse anti-rat; Proteintech) diluted 1:100, NeuN antibody (mouse anti-rat; Chemicon) diluted 1:200, SOM antibody (mouse anti-rat; Abgent) diluted 1:200 in antiserum solution 2 (1% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2), at room temperature.

    Techniques: Labeling

    Expression of P2X1 receptor-ir (green) and ICAM1-ir (red) in the mouse ileum villus. a P2X1 receptor-ir. b ICAM1-ir. c The merged image of a , b . A yellow arrow indicates a double labeling capillary-like structure. d P2X1 receptor-ir. e ICAM1-ir. f The merged image of d , e . A yellow arrow indicates a double labeling capillary-like structure. All the scale bars = 60 μm

    Journal: Purinergic Signalling

    Article Title: Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human

    doi: 10.1007/s11302-018-9615-6

    Figure Lengend Snippet: Expression of P2X1 receptor-ir (green) and ICAM1-ir (red) in the mouse ileum villus. a P2X1 receptor-ir. b ICAM1-ir. c The merged image of a , b . A yellow arrow indicates a double labeling capillary-like structure. d P2X1 receptor-ir. e ICAM1-ir. f The merged image of d , e . A yellow arrow indicates a double labeling capillary-like structure. All the scale bars = 60 μm

    Article Snippet: The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2) for 30 min, followed by incubation with different combinations of P2X1 antibody (Alomone, Israel, rabbit anti-rat) diluted 1:200, SPMA antibody (mouse anti-rat; Santa Cruz) diluted 1:200, ICAM1 antibody (mouse anti-rat; Proteintech) diluted 1:100, NeuN antibody (mouse anti-rat; Chemicon) diluted 1:200, SOM antibody (mouse anti-rat; Abgent) diluted 1:200 in antiserum solution 2 (1% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2), at room temperature.

    Techniques: Expressing, Labeling

    The results of peptide-absorptive control experiments and Western blot. a , c P2X1 receptor-ir in the villus core and myenteric plexus of mouse ileum, respectively. b , d The results of P2X1 receptor antibody and peptide-absorptive control experiments in the villus core and myenteric plexus of mouse ileum. Note that no P2X1 receptor-ir was detected in the villus core and myenteric plexus in the specimens of the peptide-absorptive control experiment. e The expression of P2X1 receptors detected by Western blotting from mouse ileum extracts. M molecular weight marker. Lane 1 is the P2X1 receptor-ir band located at about 55 kDa. Lane 2 is the preabsorption of the P2X1 receptor antisera with its peptide antigen, which resulted in the absence of the band. f is the expression of P2X1 receptors detected by Western blotting from mouse gastrointestinal tract and pancreas extracts. Lanes 1 to 5 are representative levels of P2X1 receptor-ir from the stomach corpus, jejunum, ileum, distal colon, and pancreas of the mouse. g The summary of P2X1 receptor/GAPDH ratios. The scale bars in a – d = 60 μm

    Journal: Purinergic Signalling

    Article Title: Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human

    doi: 10.1007/s11302-018-9615-6

    Figure Lengend Snippet: The results of peptide-absorptive control experiments and Western blot. a , c P2X1 receptor-ir in the villus core and myenteric plexus of mouse ileum, respectively. b , d The results of P2X1 receptor antibody and peptide-absorptive control experiments in the villus core and myenteric plexus of mouse ileum. Note that no P2X1 receptor-ir was detected in the villus core and myenteric plexus in the specimens of the peptide-absorptive control experiment. e The expression of P2X1 receptors detected by Western blotting from mouse ileum extracts. M molecular weight marker. Lane 1 is the P2X1 receptor-ir band located at about 55 kDa. Lane 2 is the preabsorption of the P2X1 receptor antisera with its peptide antigen, which resulted in the absence of the band. f is the expression of P2X1 receptors detected by Western blotting from mouse gastrointestinal tract and pancreas extracts. Lanes 1 to 5 are representative levels of P2X1 receptor-ir from the stomach corpus, jejunum, ileum, distal colon, and pancreas of the mouse. g The summary of P2X1 receptor/GAPDH ratios. The scale bars in a – d = 60 μm

    Article Snippet: The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2) for 30 min, followed by incubation with different combinations of P2X1 antibody (Alomone, Israel, rabbit anti-rat) diluted 1:200, SPMA antibody (mouse anti-rat; Santa Cruz) diluted 1:200, ICAM1 antibody (mouse anti-rat; Proteintech) diluted 1:100, NeuN antibody (mouse anti-rat; Chemicon) diluted 1:200, SOM antibody (mouse anti-rat; Abgent) diluted 1:200 in antiserum solution 2 (1% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2), at room temperature.

    Techniques: Western Blot, Expressing, Molecular Weight, Marker

    Expression of P2X1 receptors, SOM, glucagon, and insulin in mouse pancreatic islets. a P2X1 receptor-ir. b SOM-ir in the same region of a . c The merged image of a and b . Note that all the SOM-ir cells were labeled with P2X1 receptor-ir. A yellow arrow indicates a typical double-labeled cell. A green arrow indicates a small blood vessel with single labeling for P2X1 receptor-ir. d P2X1 receptor-ir. e Glucagon-ir in the same region of d . f The merged image of d and e and further immunostained with an antibody to insulin (blue color) in the same region of d and e . Note that no double or triple labeling in cells was detected. All the scale bars = 60 μm

    Journal: Purinergic Signalling

    Article Title: Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human

    doi: 10.1007/s11302-018-9615-6

    Figure Lengend Snippet: Expression of P2X1 receptors, SOM, glucagon, and insulin in mouse pancreatic islets. a P2X1 receptor-ir. b SOM-ir in the same region of a . c The merged image of a and b . Note that all the SOM-ir cells were labeled with P2X1 receptor-ir. A yellow arrow indicates a typical double-labeled cell. A green arrow indicates a small blood vessel with single labeling for P2X1 receptor-ir. d P2X1 receptor-ir. e Glucagon-ir in the same region of d . f The merged image of d and e and further immunostained with an antibody to insulin (blue color) in the same region of d and e . Note that no double or triple labeling in cells was detected. All the scale bars = 60 μm

    Article Snippet: The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2) for 30 min, followed by incubation with different combinations of P2X1 antibody (Alomone, Israel, rabbit anti-rat) diluted 1:200, SPMA antibody (mouse anti-rat; Santa Cruz) diluted 1:200, ICAM1 antibody (mouse anti-rat; Proteintech) diluted 1:100, NeuN antibody (mouse anti-rat; Chemicon) diluted 1:200, SOM antibody (mouse anti-rat; Abgent) diluted 1:200 in antiserum solution 2 (1% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2), at room temperature.

    Techniques: Expressing, Labeling

    Expression of P2X1 receptors and SOM in the myenteric plexus of the mouse gastrointestinal tract. a – d P2X1 receptor-ir in stomach corpus, jejunum, ileum and distal colon, respectively. e – h are SOM-ir in the same regions of a – d , respectively. i – l The merged images of a and e , b and f , c and g , and d and h , respectively. Note that the scattered P2X1 receptor-ir neurons were all labeled with SOM-ir. All the scale bars = 60 μm

    Journal: Purinergic Signalling

    Article Title: Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human

    doi: 10.1007/s11302-018-9615-6

    Figure Lengend Snippet: Expression of P2X1 receptors and SOM in the myenteric plexus of the mouse gastrointestinal tract. a – d P2X1 receptor-ir in stomach corpus, jejunum, ileum and distal colon, respectively. e – h are SOM-ir in the same regions of a – d , respectively. i – l The merged images of a and e , b and f , c and g , and d and h , respectively. Note that the scattered P2X1 receptor-ir neurons were all labeled with SOM-ir. All the scale bars = 60 μm

    Article Snippet: The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2) for 30 min, followed by incubation with different combinations of P2X1 antibody (Alomone, Israel, rabbit anti-rat) diluted 1:200, SPMA antibody (mouse anti-rat; Santa Cruz) diluted 1:200, ICAM1 antibody (mouse anti-rat; Proteintech) diluted 1:100, NeuN antibody (mouse anti-rat; Chemicon) diluted 1:200, SOM antibody (mouse anti-rat; Abgent) diluted 1:200 in antiserum solution 2 (1% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2), at room temperature.

    Techniques: Expressing, Labeling

    Expression of P2X1 receptor-ir ( a , d , red), SPMA-ir ( b , green), and SOM-ir ( e , green) in the mouse stomach corpus. a P2X1 receptor-ir detected in the mucosal lamina propria and blood vessels. b SPMA-labeled smooth muscle in the same region of a . Positive cells are located in the mucosal lamina propria, mucosal muscle, and blood vessels. c The merged image of a and b . A yellow arrow indicates a double-labeled vessel with both P2X1 receptor-ir and SPMA-ir (yellow in color). A green arrow indicates a single labeled mucosal muscle layer and a red arrow indicates a single labeled cell with P2X1 immunoreactivity. d P2X1 receptor-ir in the stomach corpus. e SOM-ir in the same region of d . f The merged image of d , e and an image (blue stain) of the same region counterstained by DAPI. Note that all the P2X1 receptor-ir cells were labeled with SOM-ir. The inserted figure is a higher magnification of the region indicated by a yellow arrow. ML, mucosal layer; LP, mucosal lamina propria; M, muscle layer. The scale bars in a – c = 60 μm, in d – f = 120 μm, in inserted figure = 40 μm

    Journal: Purinergic Signalling

    Article Title: Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human

    doi: 10.1007/s11302-018-9615-6

    Figure Lengend Snippet: Expression of P2X1 receptor-ir ( a , d , red), SPMA-ir ( b , green), and SOM-ir ( e , green) in the mouse stomach corpus. a P2X1 receptor-ir detected in the mucosal lamina propria and blood vessels. b SPMA-labeled smooth muscle in the same region of a . Positive cells are located in the mucosal lamina propria, mucosal muscle, and blood vessels. c The merged image of a and b . A yellow arrow indicates a double-labeled vessel with both P2X1 receptor-ir and SPMA-ir (yellow in color). A green arrow indicates a single labeled mucosal muscle layer and a red arrow indicates a single labeled cell with P2X1 immunoreactivity. d P2X1 receptor-ir in the stomach corpus. e SOM-ir in the same region of d . f The merged image of d , e and an image (blue stain) of the same region counterstained by DAPI. Note that all the P2X1 receptor-ir cells were labeled with SOM-ir. The inserted figure is a higher magnification of the region indicated by a yellow arrow. ML, mucosal layer; LP, mucosal lamina propria; M, muscle layer. The scale bars in a – c = 60 μm, in d – f = 120 μm, in inserted figure = 40 μm

    Article Snippet: The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2) for 30 min, followed by incubation with different combinations of P2X1 antibody (Alomone, Israel, rabbit anti-rat) diluted 1:200, SPMA antibody (mouse anti-rat; Santa Cruz) diluted 1:200, ICAM1 antibody (mouse anti-rat; Proteintech) diluted 1:100, NeuN antibody (mouse anti-rat; Chemicon) diluted 1:200, SOM antibody (mouse anti-rat; Abgent) diluted 1:200 in antiserum solution 2 (1% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2), at room temperature.

    Techniques: Expressing, Labeling, Staining

    Expression of P2X1 receptors and SPMA in the mouse jejunum. a P2X1 receptor-ir at low magnification. Note that strong P2X1 receptor-ir was detected in the villus core. b SPMA-ir in the same region of a . c is the merged image of a and b . d P2X1 receptor-ir in the villus core. e SPMA-ir in the same region of d . f The merged image of d and e . Note that all smooth muscle was double-labeled with P2X1 receptor-ir and SPMA-ir. The inserted image is a higher magnification of the region indicated by a yellow arrow in f . A red arrow indicates a P2X1 receptor-ir cell in the epithelium of the villus. The scale bars in a – c = 240 μm, in d – f = 60 μm, in inserted figure = 20 μm

    Journal: Purinergic Signalling

    Article Title: Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human

    doi: 10.1007/s11302-018-9615-6

    Figure Lengend Snippet: Expression of P2X1 receptors and SPMA in the mouse jejunum. a P2X1 receptor-ir at low magnification. Note that strong P2X1 receptor-ir was detected in the villus core. b SPMA-ir in the same region of a . c is the merged image of a and b . d P2X1 receptor-ir in the villus core. e SPMA-ir in the same region of d . f The merged image of d and e . Note that all smooth muscle was double-labeled with P2X1 receptor-ir and SPMA-ir. The inserted image is a higher magnification of the region indicated by a yellow arrow in f . A red arrow indicates a P2X1 receptor-ir cell in the epithelium of the villus. The scale bars in a – c = 240 μm, in d – f = 60 μm, in inserted figure = 20 μm

    Article Snippet: The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2) for 30 min, followed by incubation with different combinations of P2X1 antibody (Alomone, Israel, rabbit anti-rat) diluted 1:200, SPMA antibody (mouse anti-rat; Santa Cruz) diluted 1:200, ICAM1 antibody (mouse anti-rat; Proteintech) diluted 1:100, NeuN antibody (mouse anti-rat; Chemicon) diluted 1:200, SOM antibody (mouse anti-rat; Abgent) diluted 1:200 in antiserum solution 2 (1% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2), at room temperature.

    Techniques: Expressing, Labeling

    Expression of P2X1 receptors, SOM, glucagon, and insulin in human pancreatic islets. a P2X1 receptor-ir. b SOM-ir in the same region of a . c The merged image of a and b . Note that all the SOM-ir cells were labeled with P2X1 receptor-ir. A yellow arrow indicates a typical double-labeled cell. d P2X1 receptor-ir. e Glucagon-ir in the same region of d . f The merged image of d and e and further immunostained with an antibody to insulin (blue color) in the same region of d and e . Note that no double or triple labeling in cells was detected. All the scale bars = 60 μm

    Journal: Purinergic Signalling

    Article Title: Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human

    doi: 10.1007/s11302-018-9615-6

    Figure Lengend Snippet: Expression of P2X1 receptors, SOM, glucagon, and insulin in human pancreatic islets. a P2X1 receptor-ir. b SOM-ir in the same region of a . c The merged image of a and b . Note that all the SOM-ir cells were labeled with P2X1 receptor-ir. A yellow arrow indicates a typical double-labeled cell. d P2X1 receptor-ir. e Glucagon-ir in the same region of d . f The merged image of d and e and further immunostained with an antibody to insulin (blue color) in the same region of d and e . Note that no double or triple labeling in cells was detected. All the scale bars = 60 μm

    Article Snippet: The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2) for 30 min, followed by incubation with different combinations of P2X1 antibody (Alomone, Israel, rabbit anti-rat) diluted 1:200, SPMA antibody (mouse anti-rat; Santa Cruz) diluted 1:200, ICAM1 antibody (mouse anti-rat; Proteintech) diluted 1:100, NeuN antibody (mouse anti-rat; Chemicon) diluted 1:200, SOM antibody (mouse anti-rat; Abgent) diluted 1:200 in antiserum solution 2 (1% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2), at room temperature.

    Techniques: Expressing, Labeling

    Expression of P2X1 receptor-ir and SPMA-ir in the mouse ileum villus under confocal microscope. a P2X1 receptor-ir. b SPMA-r. c DAPI. d The merged image of a – c . An arrow indicates a typical smooth muscle fiber double labeled with P2X1 receptor-ir and SPMA-ir in d . A red arrow head indicates a capillary-like structure in d . All the scale bars = 60 μm

    Journal: Purinergic Signalling

    Article Title: Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human

    doi: 10.1007/s11302-018-9615-6

    Figure Lengend Snippet: Expression of P2X1 receptor-ir and SPMA-ir in the mouse ileum villus under confocal microscope. a P2X1 receptor-ir. b SPMA-r. c DAPI. d The merged image of a – c . An arrow indicates a typical smooth muscle fiber double labeled with P2X1 receptor-ir and SPMA-ir in d . A red arrow head indicates a capillary-like structure in d . All the scale bars = 60 μm

    Article Snippet: The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2) for 30 min, followed by incubation with different combinations of P2X1 antibody (Alomone, Israel, rabbit anti-rat) diluted 1:200, SPMA antibody (mouse anti-rat; Santa Cruz) diluted 1:200, ICAM1 antibody (mouse anti-rat; Proteintech) diluted 1:100, NeuN antibody (mouse anti-rat; Chemicon) diluted 1:200, SOM antibody (mouse anti-rat; Abgent) diluted 1:200 in antiserum solution 2 (1% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2), at room temperature.

    Techniques: Expressing, Microscopy, Labeling

    Expression of P2X1 receptors and SPMA in the mouse ileum. a – c P2X1 receptor-ir. d – f SPMA-ir. g The merged image of a and d and counterstained with DAPI. Note that strong P2X1 receptor-ir was detected in the villus core. h The merged image of b and e , counterstained with DAPI in the villus. Note that the smooth muscle was labeled with SPMA-ir and P2X1 receptor-ir. An arrow indicates a typical labeled smooth muscle fiber. i The merged image of c and f and counterstained with DAPI. A red arrow indicates a P2X1 receptor-ir cell in the gland epithelium and a red arrow head indicates a P2X1 receptor-ir cell in the submucosal layer. The scale bars in a , d , g = 240 μm, in b , c , e , f , h , i = 60 μm

    Journal: Purinergic Signalling

    Article Title: Expression of P2X1 receptors in somatostatin-containing cells in mouse gastrointestinal tract and pancreatic islets of both mouse and human

    doi: 10.1007/s11302-018-9615-6

    Figure Lengend Snippet: Expression of P2X1 receptors and SPMA in the mouse ileum. a – c P2X1 receptor-ir. d – f SPMA-ir. g The merged image of a and d and counterstained with DAPI. Note that strong P2X1 receptor-ir was detected in the villus core. h The merged image of b and e , counterstained with DAPI in the villus. Note that the smooth muscle was labeled with SPMA-ir and P2X1 receptor-ir. An arrow indicates a typical labeled smooth muscle fiber. i The merged image of c and f and counterstained with DAPI. A red arrow indicates a P2X1 receptor-ir cell in the gland epithelium and a red arrow head indicates a P2X1 receptor-ir cell in the submucosal layer. The scale bars in a , d , g = 240 μm, in b , c , e , f , h , i = 60 μm

    Article Snippet: The preparations were washed 3 × 5 min in PBS and then preincubated in antiserum solution 1 (10% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2) for 30 min, followed by incubation with different combinations of P2X1 antibody (Alomone, Israel, rabbit anti-rat) diluted 1:200, SPMA antibody (mouse anti-rat; Santa Cruz) diluted 1:200, ICAM1 antibody (mouse anti-rat; Proteintech) diluted 1:100, NeuN antibody (mouse anti-rat; Chemicon) diluted 1:200, SOM antibody (mouse anti-rat; Abgent) diluted 1:200 in antiserum solution 2 (1% normal bovine serum, 0.2% Triton X-100, 0.4% sodium azide in 0.01 mol/L PBS pH 7.2), at room temperature.

    Techniques: Expressing, Labeling

    The surface expression of platelet purinergic receptors show a biphasic variation in washed platelets after preparation. Washed platelets (3 x 10 8 per mL resting in HEPES buffer at RT) and platelets in PRP were stained with purinergic receptor specific antibodies at different time points after preparation as indicated. The basal receptor surface expression was quantified by flow cytometry. The histograms show the mean fluorescence intensities (MFI) of P2Y1 (A and D), P2Y12 (B and E) and P2X1 (C and F). Results are presented in absolute arbitrary units as mean ± SEM; n = 5; *: p

    Journal: PLoS ONE

    Article Title: Role of Purinergic Receptor Expression and Function for Reduced Responsiveness to Adenosine Diphosphate in Washed Human Platelets

    doi: 10.1371/journal.pone.0147370

    Figure Lengend Snippet: The surface expression of platelet purinergic receptors show a biphasic variation in washed platelets after preparation. Washed platelets (3 x 10 8 per mL resting in HEPES buffer at RT) and platelets in PRP were stained with purinergic receptor specific antibodies at different time points after preparation as indicated. The basal receptor surface expression was quantified by flow cytometry. The histograms show the mean fluorescence intensities (MFI) of P2Y1 (A and D), P2Y12 (B and E) and P2X1 (C and F). Results are presented in absolute arbitrary units as mean ± SEM; n = 5; *: p

    Article Snippet: Rabbit polyclonal anti-P2Y1, anti-P2Y12 and anti-P2X1 antibodies were from Alomone Labs (Jerusalem, Israel).

    Techniques: Expressing, Staining, Flow Cytometry, Cytometry, Fluorescence

    P2X1 function remains stable in washed platelets and in PRP. The figures show the calcium induced fluorescence curves in Fluo-4AM loaded washed platelets (A) and in platelets from PRP (B) after stimulation with the P2X1 agonist α,β-MeATP at different time points after preparation as indicated. Mean fluorescence curves of five independent experiments are presented (relative fluorescence units, R.F.U.).

    Journal: PLoS ONE

    Article Title: Role of Purinergic Receptor Expression and Function for Reduced Responsiveness to Adenosine Diphosphate in Washed Human Platelets

    doi: 10.1371/journal.pone.0147370

    Figure Lengend Snippet: P2X1 function remains stable in washed platelets and in PRP. The figures show the calcium induced fluorescence curves in Fluo-4AM loaded washed platelets (A) and in platelets from PRP (B) after stimulation with the P2X1 agonist α,β-MeATP at different time points after preparation as indicated. Mean fluorescence curves of five independent experiments are presented (relative fluorescence units, R.F.U.).

    Article Snippet: Rabbit polyclonal anti-P2Y1, anti-P2Y12 and anti-P2X1 antibodies were from Alomone Labs (Jerusalem, Israel).

    Techniques: Fluorescence