rabbit polyclonal anti p2y1r antibody  (Alomone Labs)

Journal: International Journal of Molecular Sciences

Article Title: P2Y 1 and P2Y 12 Receptors Mediate Aggregation of Dog and Cat Platelets: A Comparison to Human Platelets

doi: 10.3390/ijms26031206

Figure Lengend Snippet: Presence of P2Y 1 and P2Y 12 in dog, cat, and human platelets. ( A , B ) PRP was isolated from the whole blood of dogs, cats, or humans to obtain platelets. Whole lysates of platelets were analysed by immunoblotting using an ( A ) anti-P2Y 1 or ( B ) anti-P2Y 12 receptor antibody (top panels) or ( A , B ) anti-actin antibody (bottom panels). Arrows indicate ( A ) P2Y 1 or ( B ) P2Y 12 receptors. Results are representative of three independent experiments.

Article Snippet: Rabbit anti-human anti-P2Y 1 (cat. No. APR-021) and rabbit anti-human anti-P2Y 12 (cat. No. APR-020) polyclonal antibodies were from Alomone Labs (Jerusalem, Israel).

Techniques: Isolation, Western Blot

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: The P2Y 13 receptor is expressed by qNSCs in the adult SEZ. A. The expression of the different metabotropic P2Y receptors in the adult SEZ was analyzed by quantitative RT-PCR (n=6). B. P2Y 13 receptor expression in the ventral and dorsal wall of the adult SEZ analyzed by quantitative RT-PCR (n=6). C . Comparison of the P2Y 13 receptor protein in the ventral and dorsal wall of the SEZ (n=3). D-F . P2Y 13 receptor expression in the ventral wall of the SEZ. Note how the P2Y 13 receptor (red) co-localizes with GFAP positive (white) astroglia (yellow arrowheads) but not with Ascl1 positive TAPs or cells expressing βIII-tubulin (green, scale bar 30 µm except for Ascl1 10 µm). G . Co-localization of the P2Y 13 receptor (red) in NSCs with SOX2 (white) and GFAP (green) in the adult SEZ (scale bar 30 µm). H. SEZ-derived cell populations expressing the P2Y 13 receptor in a FACs analysis using a P2Y 13 -GFP conjugated antibody. Note how the expression is mainly associated with the qNSC fraction (n=3). All graphs show the mean ±SEM: *p<0.05, **p<0.01, and ***p<0.001 (T-test).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Expressing, Quantitative RT-PCR, Comparison, Derivative Assay

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Expression of the P2Y 13 receptor in the neurogenic lineage of the SEZ-derived cell cultures after 6 DIV: GFAP (magenta), Ascl1 (green, middle panel), βIII-tubulin (green, lower panel), P2Y 13 (red). Note how P2Y 13 receptor expression only co-localizes with GFAP in cells. Scale bar 30 µm.

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Expressing, Derivative Assay

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: The P2Y 13 receptor is active in SEZ-derived NSCs in culture. NSCs in culture and loaded with the calcium dye Fura-2 were stimulated with 2MeSADP and after a washout period, they were re-exposed to 2MeSADP in the presence of either the P2Y1 receptor antagonist MRS2179 A or the P2Y 13 receptor antagonist MRS2179 B . All compounds were tested at a concentration of 10 μM and representative traces of the F340/F380 fluorescence ratios recorded from single cells are shown. C . Immunocytochemistry identifying GFAP (green) and SOX2 (red) double positive NSCs that respond to the selective agonist of the P2Y 13 receptor, 2MeSADP (yellow arrowheads). Conversely, the intracellular calcium levels of GFAP positive parenchymal astrocytes (white arrow) does not change. D . Effects of 2MeSADP on voltage-gated currents in SEZ-derived NSCs in culture. Outward currents evoked by a depolarising pulse (+10 mV, 100 ms from a Vh of −80 mV) were increased in the presence of 2MeSADP (10 µM, 2 min). Subsequent exposure to MRS221 (10 µM, 2 min) partially reversed the potentiating effect of 2MeSADP (upper panel). At variance, exposure to MRS2179 (10 µM, 2 min) of 2MeSADP with MRS2179 (10 µM, 2 min) did not have any effect on outward current increase elicited by 2MeSADP (10 µM, 2 min) (lower panel). E . Scatter plot of outward current charges from the experiments shown in ( D ), in the presence or absence of 2MeSADP (10 µM, 2 min: left panel), and following co-incubation (right panels) with MRS2211 (upper right) or MRS2179 (lower right). The values are the means ± SEM of the number of cells indicated between parentheses; the statistical significance was assessed using the student’s T-test for paired samples: *p < 0.05; **p < 0.01.

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Derivative Assay, Concentration Assay, Fluorescence, Immunocytochemistry, Incubation

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: P2Y 13 silencing increases the number of NSCs remaining in the SEZ without promoting proliferation. A. The effect of local P2Y 13 receptor silencing on the NSC population, achieved using the CRISPR/Cas9 sgP2Y 13 lentiviral vectors: Cas9 (Green), SOX2 (Magenta) and GFAP (White), the cell nuclei are stained with DAPI. The lower panels show a higher magnification of the selected areas (scale bar 50 µm). B. Quantification of the Cas9 + /GFAP + /SOX2 + cells remaining in the adult SEZ (n=4). C Effect of the local silencing of the P2Y 13 receptor using the CRISPR/Cas9 sgP2Y 13 lentiviral vectors on the proliferation within the SEZ: Cas9 (Green), Ki67 (Magenta) and GFAP (White), the cell nuclei are stained with DAPI. The lower panels show a higher magnification of the selected areas (scale bar 50 µm). D . Quantification of the Cas9 + /Ki67 + cells SEZ (n=4). E . Effect of local silencing of the P2Y 13 receptor using the CRISPR/Cas9 sgP2Y 13 lentiviral vectors on the neuroblast population: Cas9 (Green), DCX (White) and the cell nuclei are stained with DAPI. The lower panels show the higher magnification of the selected areas (scale bar 50 µm). F. Quantification of the Cas9 + /DCX + cells (n=4). All graphs show mean ±SEM: ***p<0.001 (T-test).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: CRISPR, Staining

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Scheme of the strategy to generate lentiviral vectors for the local overexpression of the P2Y 13 receptor. B . Experimental design of lentiviral injection for local overexpression or silencing of the P2Y 13 receptor. C . Positive control of the local overexpression of P2Y 13 receptor. Lentiviral injection in the adult SEZ demonstrated that all LV-GFP-P2Y 13 transduced cells (Green) co-localized with P2Y 13 expression (red).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Over Expression, Injection, Positive Control, Expressing

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Local overexpression of P2Y 13 receptors augments the proportion of cells in the RMS and decreases the number of NSCs remaining in the ventral wall of the SEZ. A. Effect of P2Y 13 on SEZ dynamics, with local overexpression promoting more cells entering the RMS and less cells remaining in the SEZ (Scale bar 50 µm). B Quantification of GFP positive cells in the ventral wall of the SEZ and RMS following LV-GFP injection (n=4). C Quantification of GFP + cells in the ventral wall of the SEZ and RMS following LV-P2Y 13 -GFP injections (n=4, scale bar 50 µm). D-E. Effect of local overexpression on the remaining GFAP + (white)/SOX2 + (red)/GFP + (Green) cells in the SEZ after LV-GFP or LV-P2Y 13 -GFP injections (n=4, scale bar 50 µM). F. Quantification of GFAP + /SOX2 + /GFP + cells in the ventral wall of the SEZ following LV-GFP or LV-P2Y 13 -GFP injections (n=4). All graphs show the mean ±SEM: *p<0.05 (T-test).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Over Expression, Injection

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Transcriptomic analysis of the RNA-seq data obtained from P2Y 13 overexpressing cells in vivo . A. Scheme of the experimental design: right panel created with BioRender.com. B. A principal component analysis (PCA) of the most variably expressed (top 3,000) genes of the samples profiled, colored by condition: control ( FO_C , blue), P2Y 13 overexpression ( FO_Y , green). C. Heat-map of the top 1,000 most variably expressed genes across the samples profiled. The expression of each gene was scaled as a z-score, and the genes and sample labels were sorted by hierarchical clustering. D. Heat-map of the genes associated with the activation/quiescence equilibrium and self-renewal. The expression of each gene was scaled as a z-score, and the genes and sample labels were sorted by hierarchical clustering. E. Volcano plot showing the differential expression of genes between P2Y 13 overexpressing and control samples (n=3, PY 13 and 3 controls). Differentially expressed genes (DEGs, adjusted p-value <0.05) are in red and green when upregulated or downregulated in the P2Y 13 overexpressing samples, respectively. The bar plot represents the number of DEGs. F. Overrepresentation of gene ontology (GO) terms for biological processes from the DEGs (adjusted p-value <0.05) between the P2Y 13 overexpressing and control samples. A customized selection (18) of significant GO terms with the highest gene ratio (top 100) is displayed for clearer representation. The terms are ordered by significance, representing their adjusted p-value (x-axis), and the expressed genes were used as the background in this analysis. G. Stacked bar plot of the inferred cellular composition of neural progenitors for each condition. The cell deconvolutional analysis was carried out using CIBERSORTx, and the gene signatures of the neural progenitors were retrieved from the data generated in Belenguer et al., 2021.

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: RNA Sequencing Assay, In Vivo, Control, Over Expression, Expressing, Activation Assay, Selection, Generated

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Neurogenic trees from the ventral wall of the SEZ tracked in culture following treatment with the P2Y 13 agonist 2MesADP, in the presence or absence of the MRS2211 antagonist. A Representative symmetric neurogenic tree obtained in control conditions after 6 days in culture (N, neuron; X, cell death). B Complex symmetric neurogenic trees of 5 rounds of division obtained in the presence of 2MeSADP after 6 days in culture (N, neuron; X, cell death). The phase contrast images in both A and B depict the lineage progression in the live imaging experiment (day-hour-min). The last images show post-imaging immunocytochemistry of the neuroblast progeny (βIII-tubulin in green) Scale bar 30 µm. C Clones undergoing 1-6 rounds of division in the live imaging experiments (n=5) (n=4 for MRS2211). D Summary of all the clones tracked undergoing 4 or 5 rounds of division in our live imaging experiments, either in control conditions, or when exposed to 2MeSADP or 2MeSADP + MRS2211 (n=5) (n=4 for MRS2211). E Cell survival in the lineage trees (n=5). In all cases the progeny generated were identified by post-imaging immunocytochemistry. All graphs show the mean ±SEM: *p<0.05, and **p<0.01 (ANOVA with a Tukey’s post-test).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Control, Imaging, Immunocytochemistry, Clone Assay, Generated

Journal: bioRxiv

Article Title: Purinergic Receptor P2Y 13 Controls Activation and Mode of Division in Subependymal Adult Neural Stem Cells

doi: 10.1101/2024.11.29.626065

Figure Lengend Snippet: Effect of the P2Y 13 receptor on NSC self-renewal. A . Quantification of the symmetric lineage trees (generating only neuroblasts) and the asymmetric lineage trees (generating neuroblasts and new NSCs through self-renewal events) in control conditions, or on exposure to 2MeSADP or 2MeSADP + MRS2211 (n=4). B. Proportion of GFAP + /SOX2 + NSCs relative to the total number of cells in the culture according to the following labels: “Initial” indicates the proportion of GFAP + /SOX2 + in the culture and the “Quiescent” cells are the fraction of the initial cells that remain quiescent throughout the live imaging experiments. The “Clones” reflect the fraction of the initial cells that undergo lineage progression (Control and 2MeSADP n=5 and 2MeSADP+MRS2211 n = 4). C. The models represent the stereotypic behavior of NSCs under control conditions and when exposed to 2MeSADP or 2MeSADP+MRS211. Note how 2MeSADP increases the speed in the cell cycle while impeding self-renewal divisions. By contrast, MRS2211 instructs NSCs to remain quiescent. D. Cell Cycle length within the complex trees tracked (≥4 rounds of division). E . Cell Cycle length for the first division within the complex trees tracked (≥4 rounds of division, n= 5). Note that this first division is normally associated with slow-dividing astroglia, yet 2MeSADP significantly increase the speed of the cell cycle (n=5). All the graphs show the mean ±SEM: *p<0.05, **p<0.01 (T-test for D, E and ANOVA with a Tukeys post-test for B ).

Article Snippet: Both the cells and tissue sections were then incubated overnight at 4 °C with the primary antibodies: rabbit anti-P2Y 13 (1:100, Alomone Labs Cat# APR-009), rabbit anti-P2Y 1 (1:100, Alomone Labs Cat# APR-017), rabbit anti-SOX2 (1:100, ABclonal Cat# A0561), mouse anti-GFAP (1:200, Sigma-Aldrich Cat# G3893), mouse anti-βIII-Tubulin (1:800, Sigma-Aldrich Cat# T8660), guinea pig anti-(Doublecortin)DCX (1:400, Millipore Cat# AB2253), mouse anti-ASCL1 (1:100, BD Biosciences Cat# 556604), rabbit anti-KI67 (1:100, Fisher Scientific Cat# RM-9106-S), and chicken anti-GFP (1:400, AvesLab Cat# GFP-1020).

Techniques: Control, Imaging, Clone Assay

Journal: Molecular Metabolism

Article Title: Exploring the role of purinergic receptor P2RY1 in type 2 diabetes risk and pathophysiology: Insights from human functional genomics

doi: 10.1016/j.molmet.2023.101867

Figure Lengend Snippet: Location of the variants found in P2RY1 . Schematic representation of the human form of the P2RY1 receptor (encoded by NM_002563.5) according to Swiss-Prot (via the GPCR database [ https://gpcrdb.org/ ]). Red represents variant inducing amino acid changes linked to loss-of-function variants, while blue represents variant inducing amino acid changes linked to neutral variants according to our in vitro assays. C-term , Carboxyl-Terminus; ECL , Extra Cellular Loop; ICL , Intra Cellular Loop; N-term , Amino-Terminus. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The human P2RY1 (NM_002563.5) plasmid was purchased from Origene (Rockville, USA).

Techniques: Variant Assay, In Vitro

Journal: Molecular Metabolism

Article Title: Exploring the role of purinergic receptor P2RY1 in type 2 diabetes risk and pathophysiology: Insights from human functional genomics

doi: 10.1016/j.molmet.2023.101867

Figure Lengend Snippet: In vitro analyses for characterizing the activity of loss-of-function P2RY1 variants in the NFAT-RE pathway . NFAT-RE luciferase activation was shown following (A) dose–response (during a 6-hour period) and (B) time course (at 10 uM concentration) experiments for each P2RY1 loss-of-function variant. Each mutant (depicted by black triangles) was compared to the activity of wild-type P2RY1 (positive control shown as blue dots) and a P2RY1 mutant harboring an early stop-codon variant (negative control represented by a red square). Data are presented as percentages of wild-type maximum activity. The results represent the means ± SEM of 4 independent experiments, each performed in technical quadruplicate. (C) Radar charts represent signaling signature of the seven loss-of-function variants (represented in black) and wild-type P2RY1 (shown in blue). Parameters measured include protein expression, basal activity, maximum effect upon agonist stimulation (Max), gap between minimum and maximum effects (Span), half maximal effective concentration (EC50), and activity over time (Hillslope). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The human P2RY1 (NM_002563.5) plasmid was purchased from Origene (Rockville, USA).

Techniques: In Vitro, Activity Assay, Luciferase, Activation Assay, Concentration Assay, Variant Assay, Mutagenesis, Positive Control, Negative Control, Expressing

Journal: Molecular Metabolism

Article Title: Exploring the role of purinergic receptor P2RY1 in type 2 diabetes risk and pathophysiology: Insights from human functional genomics

doi: 10.1016/j.molmet.2023.101867

Figure Lengend Snippet: Negative association between P2RY1 expression levels in human pancreatic islets and T2D risk alleles . On the X-axis, the localization of expression quantitative trait loci (eQTLs) at the P2RY1 locus is represented, while the Y-axis represents the p -value of their association with T2D risk. Blue up-pointing triangles represent SNPs significantly associated with increased P2RY1 expression in human pancreatic islets and reduced T2D risk (with a p-value <0.05), while red down-pointing triangles represent SNPs significantly associated with decreased P2RY1 expression in islets and increased T2D risk (with a p-value <0.05). The dotted line indicates the multiple testing threshold. Below the X-axis, a chromatin map from human pancreatic beta-cell line EndoCβH1 is represented. The chromatin elements are color-coded as follows: light green for weak enhancers, dark green for active enhancers, yellow for active transcription start sites (TSS), and blue for transcription sites. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The human P2RY1 (NM_002563.5) plasmid was purchased from Origene (Rockville, USA).

Techniques: Expressing

Journal: Molecular Metabolism

Article Title: Exploring the role of purinergic receptor P2RY1 in type 2 diabetes risk and pathophysiology: Insights from human functional genomics

doi: 10.1016/j.molmet.2023.101867

Figure Lengend Snippet: P2RY1 expression in human pancreatic beta cells and P2RY1 effect on glucose-stimulated insulin secretion upon its stimulation or inhibition . (A) Expression of P2RY1 in human purified pancreatic beta cells ( n = 11) and non-beta cell (remaining islet preparation) ( n = 5) assessed via . Data are mean ± SEM of RPKM (Reads Per Kilobase Million). (B) Expression of P2RY1 in Fetal and Adult human beta and alpha cells (between 6 and 7 samples per condition) assessed via . Data are mean ± SEM of TPM (Transcripts Per Million). Glucose-stimulated insulin secretion (glucose [G] 0 mM versus [G] 20 mM) from EndoCβH5 treated with (C) ± 1 μM of P2RY1-specific agonist MRS2365; or (D) ± 1 μM of P2RY1-specific antagonist MRS2500. (E) Potassium-chloride stimulated insulin secretion ([KCl] 0 mM versus [KCl] 25 mM) from EndoCβH5 treated with ±1 μM of P2RY1-specific agonist MRS2365. (F) Glucose stimulated insulin secretion (glucose [G] 0 mM versus [G] 20 mM) from EndoCβH5 treated with ±1 μM Diazoxide ± 1 μM of P2RY1-specific agonist MRS2365. Data are the means ± SEM from three independent experiments, each conducted in technical quadruplicate. All experiments were analyzed using a Mann–Whitney t -test: ∗ P < 0.01; ∗∗ P < 0.01; ∗∗∗ P < 0.001. CT , control condition.

Article Snippet: The human P2RY1 (NM_002563.5) plasmid was purchased from Origene (Rockville, USA).

Techniques: Expressing, Inhibition, Purification, MANN-WHITNEY, Control

Journal: Frontiers in Immunology

Article Title: SARS-CoV-2 Spike protein alters microglial purinergic signaling

doi: 10.3389/fimmu.2023.1158460

Figure Lengend Snippet: SARS−CoV−2 spike protein increases ATP secretion and purinergic signaling components transcript levels in microglial cells. BV-2 cells were left untreated (Ctrl) or stimulated with 0.5 or 1 µg/mL SARS−CoV−2 spike protein for 2h for ATP quantification in culture supernatants or 24 h for qPCRs. (A) ATP concentration in cell supernatants. The levels of transcripts for (B) P2X7, (C) P2X4, (D) P2Y 1 , (E) P2Y 2 , (F) P2Y 4 , (G) P2Y 6 , (H) P2Y 12 , (I) E-NTPDase1, (J) E-NTPDase2, (K) E-NTPDase3, and (L) CD73 were analyzed by RT-qPCR. Data are representative of three independent experiments (n=3) performed in triplicates and expressed as mean ± SEM. Statistically significant differences between Ctrl and treated groups are represented by asterisks (*, p < 0.05). One-way analysis of variance, Tukey’s test.

Article Snippet: Samples were then incubated overnight with the following primary antibodies diluted in 0.1% BSA in PBS: rabbit anti-P2X7 #APR-008 (1:100), rabbit anti-P2Y 1 #APR-009 (1:100), rabbit anti-P2Y 4 #APR-006 (1:300), rabbit anti-P2Y 6 #APR-011 (1:500), rabbit anti-P2Y 12 #APR-012 (1:50) (Alomone Labs, Jerusalem, Israel), goat anti-CD39 #AF4398 (1:200) (R&D Systems, Minneapolis, MN), rabbit ENTPD2/CD39L1 # BS-11515R (1:100) (Thermo Fisher Scientific, NJ, USA).

Techniques: Concentration Assay, Quantitative RT-PCR

Journal: Frontiers in Immunology

Article Title: SARS-CoV-2 Spike protein alters microglial purinergic signaling

doi: 10.3389/fimmu.2023.1158460

Figure Lengend Snippet: SARS−CoV−2 spike protein increases the expression of P2 receptors and ectonucleotidases in microglial cells. BV-2 cells were left untreated (Ctrl) or stimulated with 1 µg/mL SARS−CoV−2 spike protein for 24 h. Representative images and quantitative analysis for (A–C) P2X7, (D–F) P2Y 1 , (G–I) P2Y 4 , (J–L) P2Y 6 , (M–O) P2Y 12 , E-NTPDase1 (P–R) , and E-NTPDase2 (S–U) . Data are representative of three independent experiments (n=3) and expressed as mean ± SEM of 10 fields per condition. (V) ATP hydrolysis assay. Data are representative of three independent experiments (n=3) and expressed as mean ± SEM. Statistically significant differences between Ctrl and treated groups are represented by asterisks (*p < 0.05; **p < 0.01; ****p < 0.0001). Student’s t-test. Scale bars: 20 μm.

Article Snippet: Samples were then incubated overnight with the following primary antibodies diluted in 0.1% BSA in PBS: rabbit anti-P2X7 #APR-008 (1:100), rabbit anti-P2Y 1 #APR-009 (1:100), rabbit anti-P2Y 4 #APR-006 (1:300), rabbit anti-P2Y 6 #APR-011 (1:500), rabbit anti-P2Y 12 #APR-012 (1:50) (Alomone Labs, Jerusalem, Israel), goat anti-CD39 #AF4398 (1:200) (R&D Systems, Minneapolis, MN), rabbit ENTPD2/CD39L1 # BS-11515R (1:100) (Thermo Fisher Scientific, NJ, USA).

Techniques: Expressing, Hydrolysis Assay

Journal: Frontiers in Immunology

Article Title: SARS-CoV-2 Spike protein alters microglial purinergic signaling

doi: 10.3389/fimmu.2023.1158460

Figure Lengend Snippet: SARS−CoV−2 spike protein increases purinergic signaling components transcript levels in mice hippocampus. The levels of transcripts for (A) P2X7, (B) P2X4, (C) P2Y 1 , (D) P2Y 2 , (E) P2Y 4 , (F) P2Y 6 , (G) P2Y 12 , (H) E-NTPDase1, (I) E-NTPDase2, (J) E-NTPDase3, and (K) CD73 in hippocampus of mice post-spike ICV injection were analyzed by RT-qPCR. Data are expressed as mean ± SEM. Statistically significant differences between the control (Ctrl) and treated group are represented by asterisks (*, p < 0.05). Outliers were identified and excluded using Prism 8.0.1 software (GraphPad Software, La Jolla, CA, USA). Student’s t-test (Control group n = 3; Spike group n = 5–7).

Article Snippet: Samples were then incubated overnight with the following primary antibodies diluted in 0.1% BSA in PBS: rabbit anti-P2X7 #APR-008 (1:100), rabbit anti-P2Y 1 #APR-009 (1:100), rabbit anti-P2Y 4 #APR-006 (1:300), rabbit anti-P2Y 6 #APR-011 (1:500), rabbit anti-P2Y 12 #APR-012 (1:50) (Alomone Labs, Jerusalem, Israel), goat anti-CD39 #AF4398 (1:200) (R&D Systems, Minneapolis, MN), rabbit ENTPD2/CD39L1 # BS-11515R (1:100) (Thermo Fisher Scientific, NJ, USA).

Techniques: Injection, Quantitative RT-PCR, Control, Software