anti-p2y2 receptor antibody  (Alomone Labs)

Journal: American journal of physiology. Regulatory, integrative and comparative physiology

Article Title: GPER1 Activation Regulates Renal Purinergic P2Y 2 Receptor Natriuretic Pathway

doi: 10.1152/ajpregu.00123.2025

Figure Lengend Snippet: Relative mRNA expression of Cx30 (A-C), P2Y 2 receptor (D-F), and αENaC (G-I) in the renal cortex, outer medulla, and inner medulla from normal salt- and high salt-fed OVX SD rats treated with vehicle (Veh) or the GPER1 agonist, (G1, 400 μg/kg/day). Gene expression values represent fold change from corresponding Veh-treated group. n=5-7 in each group. Statistical comparisons performed by unpaired Student t test. Abbreviations: Cx30: connexin 30; αENaC: epithelial Na + channel alpha subunit; GPER1: G protein-coupled estrogen receptor; OVX: ovariectomized; SD: Sprague Dawley; SE: standard error of the mean; Veh: vehicle.

Article Snippet: Interestingly, ovariectomy downregulates renal P2Y 2 receptor expression in Sprague Dawley (SD) rats.

Techniques: Activation Assay, Expressing, Gene Expression

Journal: American journal of physiology. Regulatory, integrative and comparative physiology

Article Title: GPER1 Activation Regulates Renal Purinergic P2Y 2 Receptor Natriuretic Pathway

doi: 10.1152/ajpregu.00123.2025

Figure Lengend Snippet: Relative mRNA expression of Cx30 (A), P2Y 2 receptor (B), and αENaC (C) in kidneys from female wild-type (WT) and GPER1 knockout (KO) mice fed a normal salt diet. Gene expression values represent fold change from the corresponding WT group. n=7-10 in each group. Statistical comparisons were performed by unpaired Student t test. Abbreviations: Cx30: connexin 30; αENaC: epithelial Na + channel alpha subunit; GPER1: G protein-coupled estrogen receptor; KO: knockout; SE: standard error of the mean; WT: wild-type.

Article Snippet: Interestingly, ovariectomy downregulates renal P2Y 2 receptor expression in Sprague Dawley (SD) rats.

Techniques: Expressing, Knock-Out, Gene Expression

Journal: The Journal of Physiology

Article Title: Age‐related changes in P2Y receptor signalling in mouse cochlear supporting cells

doi: 10.1113/JP284980

Figure Lengend Snippet: A , diagram showing a cross‐section side‐view of a pre‐hearing (left) and mature (right) organ of Corti. IHC: inner hair cell; OHC: outer hair cell; IPhC: inner phalangeal cell; IBC: inner border cell; PC: pillar cell; GER: greater epithelial ridge; IS: inner sulcus; SC: supporting cells of the GER and IS, which also include IPhCs and IBCs. The ‘Immature’ and ‘Mature’ designations refer to before and after the onset or hearing, respectively, which in mice occurs at around P12. The images below represent either the top view (red arrow) or the side view of the GER and IS. The image on the left was modified from Ceriani et al. . B and C , brightfield images of the organ of Corti in P7 (top), 1‐month‐old (middle) and aged (bottom) 6N ( B ) and C3H ( C ) mice taken from the top‐view orientation used for the fluorescence images in panels D and E ; these images highlight the location of the different supporting cell types (SC) in the GER and IS and the IHCs. Note that for simplicity, only a few SCs are highlighted in panels B and C . Scale bars are 20 μm. D and E , maximum intensity projections of confocal z ‐stacks showing images of the GER and IS viewed from the top (red arrow in panel A ) in P7 (top panels), 1‐month‐old (middle panels) and aged (17–21 months: bottom panels) 6N ( D ) and C3H ( E ) mice. Left columns show the actin‐marker phalloidin (magenta), which is labelling the hair bundles of the IHCs (yellow arrows) and the membrane of some of the supporting cells. Middle columns show the P2Y 1 puncta‐like labelling (white) in the supporting cells from the bulk of the GER and IS (white arrows). P2Y 1 was also expressed in the pillar cells (PCs: orange arrows) which were not investigated in this study. Right panels show the merged images. Scale bars are 20 μm. F and G , maximum intensity projections of confocal z‐stack images of the GER and IS viewed from the side (see panel A ). Phalloidin: magenta; P2Y 1 : white. Note that phalloidin primarily labels the hair bundle of the IHCs (yellow circles) and the pillar cells (PC). Scale bars are 10 μm.

Article Snippet: The primary antibodies used included anti‐P2Y 1 (Alomone Labs, cat. no. APR‐009), anti‐P2Y 2 (Alomone Labs, cat. no. APR‐010) and anti‐P2Y 4 (Alomone Labs, cat. no. APR‐006) all at a concentration of 1:800.

Techniques: Modification, Fluorescence, Marker, Membrane

Journal: The Journal of Physiology

Article Title: Age‐related changes in P2Y receptor signalling in mouse cochlear supporting cells

doi: 10.1113/JP284980

Figure Lengend Snippet: A and B , maximum intensity projections of confocal z ‐stacks showing images of the GER and IS viewed from the top (red arrow in panel 2 A ) in P7 (top panels), 1‐month‐old (middle panels) and aged (17–21 months: bottom panels) 6N ( A ) and C3H ( B ) mice. Left column: actin‐marker phalloidin (magenta); middle column: P2Y 4 (white). Right panels show the merged images. For the identification of the cellular organization and labels, see Fig. . Scale bars are 20 μm. C and D , maximum intensity projections of confocal z ‐stack images of the GER and IS viewed from the side (see Fig. A ). Phalloidin: magenta; P2Y 4 : white. Scale bars are 10 μm.

Article Snippet: The primary antibodies used included anti‐P2Y 1 (Alomone Labs, cat. no. APR‐009), anti‐P2Y 2 (Alomone Labs, cat. no. APR‐010) and anti‐P2Y 4 (Alomone Labs, cat. no. APR‐006) all at a concentration of 1:800.

Techniques: Marker

Journal: The Journal of Physiology

Article Title: Age‐related changes in P2Y receptor signalling in mouse cochlear supporting cells

doi: 10.1113/JP284980

Figure Lengend Snippet: A and B , maximum intensity projections of confocal z ‐stacks showing images of the GER and IS viewed from the top (red arrow in panel 2 A ) in P7 (top panels), 1‐month‐old (middle panels) and aged (17–21 months: bottom panels) 6N ( A ) and C3H ( B ) mice. Left column: actin‐marker phalloidin (magenta); middle column: P2Y (white). Right panels show the merged images. For the identification of the cellular organisation and labels, see Fig. . Scale bars are 20 μm. C and D , maximum intensity projections of confocal z ‐stack images of the GER and IS viewed from the side (see Fig. A ). Phalloidin: magenta; P2Y 4 : white. Scale bars are 10 μm.

Article Snippet: The primary antibodies used included anti‐P2Y 1 (Alomone Labs, cat. no. APR‐009), anti‐P2Y 2 (Alomone Labs, cat. no. APR‐010) and anti‐P2Y 4 (Alomone Labs, cat. no. APR‐006) all at a concentration of 1:800.

Techniques: Marker

Journal: The Journal of Physiology

Article Title: Age‐related changes in P2Y receptor signalling in mouse cochlear supporting cells

doi: 10.1113/JP284980

Figure Lengend Snippet: A–C , representative Ca 2+ responses in supporting cells induced by the extracellular application of 1 μm ADP (grey area) in 6N mice at different age ranges shown above the recordings. D , comparison of the average Ca 2+ response at the onset of ADP application (grey bar beneath the traces) in cochlear supporting cells of 6N mice in the three different age ranges tested. Continuous traces represent averages, while the shaded area is the SD. Numbers of individual supporting cells (ROIs): P7–P8, 59 ROIs (3 mice); 1–2 months old, 70 ROIs (4), and 18–24 months old, 121 ROIs (5). E and F , comparison of the maximum ( E ) and average ( F ) Ca 2+ response to 1 μm ADP application in 6N mice at different ages. Number of supporting cells used is shown above the averages (± SD) and single data points (plotted as open circles). G , representative Ca 2+ responses in supporting cells induced by 1 μm extracellular ADP (grey area) in aged 6N mice. The application of ADP together with the P2Y 1 antagonist MRS2500 (1 μm, top black horizontal line) blocked the ADP‐induced Ca 2+ response. H , effect of P2Y 1 antagonist MRS2500 on the size of the ADP‐induced Ca 2+ response in cochlear supporting cells from aged mice from 64 supporting cells (ROIs) from 3 mice. Significance values are indicated by the asterisks ( P < 0.0001, Wilcoxon signed‐rank test).

Article Snippet: The primary antibodies used included anti‐P2Y 1 (Alomone Labs, cat. no. APR‐009), anti‐P2Y 2 (Alomone Labs, cat. no. APR‐010) and anti‐P2Y 4 (Alomone Labs, cat. no. APR‐006) all at a concentration of 1:800.

Techniques: Comparison

Journal: The Journal of Physiology

Article Title: Age‐related changes in P2Y receptor signalling in mouse cochlear supporting cells

doi: 10.1113/JP284980

Figure Lengend Snippet: A , representative Ca 2+ responses in supporting cells induced by the extracellular application of 300 n m UTP (grey area) in aged 6N mice. The application of UTP together with the P2Y 2 antagonist ARC‐118925XX (15 μm, top black horizontal line) caused a reduction of the UTP‐induced Ca 2+ response and stopped Ca 2+ oscillations. B , average UTP‐induced Ca 2+ responses during the last 10 s of UTP application and when UTP was applied together with the P2Y 2 antagonist ARC‐118925XX in cochlear supporting cells from aged mice (74 ROIs from 4 mice). Significance values are indicated by the asterisks ( P < 0.0001, Wilcoxon signed‐rank test). C , representative Ca 2+ responses in supporting cells induced by the P2Y 4 agonist MRS4062 (10 μm, top black horizontal line) in aged 6N mice. This initial response was followed by the application of 300 n m UTP alone (grey area), to confirm that the supporting cell was responsive to UTP. D , average and maximum MRS4062‐induced Ca 2+ response in cochlear supporting cells from aged mice. Open symbols are measurements from individual supporting cells (ROIs): 84 from 3 mice.

Article Snippet: The primary antibodies used included anti‐P2Y 1 (Alomone Labs, cat. no. APR‐009), anti‐P2Y 2 (Alomone Labs, cat. no. APR‐010) and anti‐P2Y 4 (Alomone Labs, cat. no. APR‐006) all at a concentration of 1:800.

Techniques:

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) RNAscope in-situ hybridization of P2Y 2 mRNA expression (magenta) in tumor and matching normal adjacent tissue. ( B ) P2Y 2 mRNA expression in tumor (TCGA) and normal (GTEx) pancreatic tissue samples (*p<0.0001). Graph generated using GEPIA. ( C ) Kaplan-Meier plot comparing patients with high vs low expression of P2Y 2 in the Pancreatic Adenocarcinoma (PAAD) The Cancer Genome Atlas (TCGA) cohort. Graph generated using KMplot. ( D ) Top result of a GSEA (performed with WebGestalt) of two different pancreatic adenocarcinoma patient cohorts (PAAD TCGA and PDAC CPTAC) for the PANTHER pathway functional database. ( E ) Incucyte images of the pancreatic cancer cell line AsPC-1 12 hr after treatment with 100 µM ATP alone or with 5 µM AR-C (P2Y 2 antagonist). Cells are transduced with Lifeact to visualize f-actin (green). ( F ) Schematic of the amino acid sequence of P2Y 2 showing an RGD motif in the first extracellular loop (image generated in http://gpcrdb.org/ ). ( G ) IF staining of P2Y 2 (green), integrin αV (red), and DAPI (blue) in AsPC-1 cells showing colocalization of P2Y 2 and integrin αV (yellow).

Article Snippet: Following blocking with 5% milk (Sigma) in 0.1% TBS-T for 1 hr, membranes were incubated with 1:1000 dilution of antibodies against phosphorylated FAK (Tyr397, 3283, Cell Signaling, RRID: AB_2173659 ), phosphorylated ERK 1/2 (S217/221, 9154, Cell Signaling, RRID: AB_2138017 ), P2Y 2 (APR-010, Alomone Labs, RRID: AB_2040078 ), HSC 70 (SC7298, Santa Cruz, RRID: AB_627761 ), or α-tubulin (T5168, Sigma-Aldrich, RRID: AB_477579 ) with 5% BSA in 0.1% TBS-T overnight at 4 °C.

Techniques: RNAscope, In Situ Hybridization, Expressing, Generated, Functional Assay, Transduction, Sequencing, Staining

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) RNAscope in-situ hybridization of a positive control ( PPIB , Cyclophilin B), negative control ( DapB ), and P2Y 2 mRNA expression in a PDAC tissue slide showing tumor and normal adjacent tissue. ( B ) Single-cell expression of P2Y 2 in healthy pancreatic tissue from the Human Protein Atlas ( https://www.proteinatlas.org/ENSG00000175591-P2RY2/single+cell+type/pancreas ). ( C ) Top four results of a GSEA (performed with WebGestalt) of two different pancreatic adenocarcinoma patient cohorts (PAAD TCGA and PDAC CPTAC) for the ‘Molecular Function’ Gene Ontology (GO) functional database. ( D ) Incucyte analysis of average object area related to the average cell area of AsPC-1 cells at different concentrations of ATP (error bars show standard deviation). ( E ) Incucyte images of AsPC-1 cells with different concentrations of AR-C with or without ATP. ( F ) IF staining of four different PDAC cell lines showing various levels of P2Y 2 (green) and integrin αV (red) protein expression. ( G ) The respective reads per kilobase of exon per million reads mapped (RPKM) from CCLE.

Article Snippet: Following blocking with 5% milk (Sigma) in 0.1% TBS-T for 1 hr, membranes were incubated with 1:1000 dilution of antibodies against phosphorylated FAK (Tyr397, 3283, Cell Signaling, RRID: AB_2173659 ), phosphorylated ERK 1/2 (S217/221, 9154, Cell Signaling, RRID: AB_2138017 ), P2Y 2 (APR-010, Alomone Labs, RRID: AB_2040078 ), HSC 70 (SC7298, Santa Cruz, RRID: AB_627761 ), or α-tubulin (T5168, Sigma-Aldrich, RRID: AB_477579 ) with 5% BSA in 0.1% TBS-T overnight at 4 °C.

Techniques: RNAscope, In Situ Hybridization, Positive Control, Negative Control, Expressing, Functional Assay, Standard Deviation, Staining

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) Schematic diagram of the hanging drop sphere model for 3D sphere invasion assays. ( B ) Bright field and fluorescent images of spheres formed using AsPC-1 cells (magenta) with a histone 2B (H2B) tagged with a red fluorescent protein (RFP) and the stellate cell line PS-1 (green) with H2B tagged with a green fluorescent protein (GFP). Middle panel shows AsPC-1 cells in spheres with a dotted line highlighting the central sphere area. Spheres were treated with vehicle control or 100 µM ATP alone or with 5 µM AR-C or 10 µM cRGDfV. The quantification is shown in ( C ) using SuperPlots, where each color represents a biological repeat (n=3) and the larger points represent the mean % Invasion for each repeat. ( D ) Quantification of spheres formed by AsPC-1 cells transfected with a control siRNA or P2Y 2 siRNA and treated with or without 100 µM ATP. ( E ) Bright field and fluorescent images of spheres formed by AsPC-1 cells subjected to CRISPR/Cas9 gene disruption using a control guide RNA (CTR CRISPR ) or P2Y 2 guide RNAs (P2Y 2 CRISPR ) and treated with or without 100 µM ATP. Quantification in ( F ). ( G, I ) Bright field and fluorescent images of AsPC-1 P2Y 2 CRISPR cells or PANC-1 cells (respectively) transfected with wild-type P2RY2 (P2Y 2 RGD ) or mutant P2RY2 D97E (P2Y 2 RGE ) treated with or without 100 µM ATP and its quantification in ( H ) and ( J ), respectively. Statistical analysis with Kuskal-Wallis multiple comparison tests.

Article Snippet: Following blocking with 5% milk (Sigma) in 0.1% TBS-T for 1 hr, membranes were incubated with 1:1000 dilution of antibodies against phosphorylated FAK (Tyr397, 3283, Cell Signaling, RRID: AB_2173659 ), phosphorylated ERK 1/2 (S217/221, 9154, Cell Signaling, RRID: AB_2138017 ), P2Y 2 (APR-010, Alomone Labs, RRID: AB_2040078 ), HSC 70 (SC7298, Santa Cruz, RRID: AB_627761 ), or α-tubulin (T5168, Sigma-Aldrich, RRID: AB_477579 ) with 5% BSA in 0.1% TBS-T overnight at 4 °C.

Techniques: Control, Transfection, CRISPR, Disruption, Mutagenesis, Comparison

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) Hanging drop sphere with and without PS-1 cells. ( B, C ) Quantification of AsPC-1 spheres treated with 100 µM UTP or ATPγS (respectively) in the absence or together with 5 µM AR-C or 10 µM cRGDfV (n=3 biological replicates). ( D ) IF staining of P2Y 2 in AsPC-1 and PS-1 stellate cells. ( E ) Migration assay with AsPC-1 and 100 µM ATP in the absence or together with 5 µM AR-C or/and 10 µM cRGDfV and ( F ) its quantification (n=3 biological replicates, statistical analysis with one-way ANOVA, * p < 0.5, **** p < 0.0001, error bars show standard deviation). ( G ) 3D sphere invasion assay using BxPC-3 cells treated with 100 µM of ATP in the absence or together with 5 µM AR-C or/and 10 µM cRGDfV and ( H ) its quantification (n=3 biological replicates). ( I ) qPCR of P2Y 2 expression and western blot of siRNA treated cells (control siRNA and P2Y 2 targeting siRNA, n=3 biological replicates). ( J ) AsPC-1 P2Y 2 CRISPR spheres treated with or without 5 µM of AR-C (n=3 biological replicates). ( K ) P2Y 2 IF staining of AsPC-1 P2Y 2 CRISPR cells transfected with an empty vector, P2Y 2 RGD or P2Y 2 RGE plasmids. Statistical analysis of all invasion experiments with Kuskal-Wallis multiple comparison tests. Figure 3—figure supplement 1—source data 1. Labeled uncropped blot of . Figure 3—figure supplement 1—source data 2. Full unedited blot of .

Article Snippet: Following blocking with 5% milk (Sigma) in 0.1% TBS-T for 1 hr, membranes were incubated with 1:1000 dilution of antibodies against phosphorylated FAK (Tyr397, 3283, Cell Signaling, RRID: AB_2173659 ), phosphorylated ERK 1/2 (S217/221, 9154, Cell Signaling, RRID: AB_2138017 ), P2Y 2 (APR-010, Alomone Labs, RRID: AB_2040078 ), HSC 70 (SC7298, Santa Cruz, RRID: AB_627761 ), or α-tubulin (T5168, Sigma-Aldrich, RRID: AB_477579 ) with 5% BSA in 0.1% TBS-T overnight at 4 °C.

Techniques: Staining, Migration, Standard Deviation, Invasion Assay, Expressing, Western Blot, Control, CRISPR, Transfection, Plasmid Preparation, Comparison, Labeling

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A, B ) Overview of the DNA-PAINT microscopy technique and qPAINT analysis pipeline. ( C ) Histogram of qPAINT indices for αV (blue) and P2Y 2 (red) single-molecule localization clusters. Solid lines represent multi-peak Gaussian fit. ( D ) Rendered DNA-PAINT images of AsPC-1 P2Y 2 CRISPR cells transfected with P2Y 2 RGD or P2Y 2 RGE with or without 100 µM of ATP and close-ups showing the protein maps reconstructed from DNA-PAINT localization maps of P2Y 2 (red) and integrin αV (cyan). The quantification of the number of proteins or protein clusters (>3 proteins) in each region of interest (ROI) are for P2Y 2 (red) ( E ) and ( F ), respectively and integrin αV (cyan) ( G ) and ( H ), respectively. Quantification of protein proximity using the nearest neighbor distance (NND), with the percentages of integrin αV and P2Y 2 proteins being <50 nm apart ( I ), between different αV integrins being 20–100 nm ( J ) or <20 nm ( K ) apart; and P2Y 2 from other P2Y 2 proteins being <40 nm apart ( H ). Statistical analysis with Kuskal-Wallis multiple comparison test of 21 4x4 µm ROIs from a minimum of 5 cell regions per condition.

Article Snippet: Following blocking with 5% milk (Sigma) in 0.1% TBS-T for 1 hr, membranes were incubated with 1:1000 dilution of antibodies against phosphorylated FAK (Tyr397, 3283, Cell Signaling, RRID: AB_2173659 ), phosphorylated ERK 1/2 (S217/221, 9154, Cell Signaling, RRID: AB_2138017 ), P2Y 2 (APR-010, Alomone Labs, RRID: AB_2040078 ), HSC 70 (SC7298, Santa Cruz, RRID: AB_627761 ), or α-tubulin (T5168, Sigma-Aldrich, RRID: AB_477579 ) with 5% BSA in 0.1% TBS-T overnight at 4 °C.

Techniques: Microscopy, CRISPR, Transfection, Comparison

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) Schematic diagram of the predicted maximum distance between fluorescent molecules indicating physical contact between proteins, to the nearest first significant figure. ( B ) Histograms of the nearest neighbor distance between proteins vs the frequency of occurrence for AsPC-1 P2Y 2 CRISPR in different conditions (solid line, strong color) or randomly computer-generated controls (dotted line, light color).

Article Snippet: Following blocking with 5% milk (Sigma) in 0.1% TBS-T for 1 hr, membranes were incubated with 1:1000 dilution of antibodies against phosphorylated FAK (Tyr397, 3283, Cell Signaling, RRID: AB_2173659 ), phosphorylated ERK 1/2 (S217/221, 9154, Cell Signaling, RRID: AB_2138017 ), P2Y 2 (APR-010, Alomone Labs, RRID: AB_2040078 ), HSC 70 (SC7298, Santa Cruz, RRID: AB_627761 ), or α-tubulin (T5168, Sigma-Aldrich, RRID: AB_477579 ) with 5% BSA in 0.1% TBS-T overnight at 4 °C.

Techniques: CRISPR, Generated

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A, B ) Western blots of phosphorylated FAK (p-FAK) and ERK (p-ERK) of AsPC-1 cells treated with ATP or pre-treated for 30 min with AR-C (5 µM) or cRGDfV (10 µM), respectively and treated with ATP for 60 min. ( C ) Western blot of AsPC-1 P2Y 2 CRISPR cells transfected with P2Y 2 RGD or P2Y 2 RGE and treated with ATP for 60 min. Representative images of three biological replicates. Figure 5—source data 1. Labeled uncropped blots of . Figure 5—source data 2. Full unedited blots of .

Article Snippet: Following blocking with 5% milk (Sigma) in 0.1% TBS-T for 1 hr, membranes were incubated with 1:1000 dilution of antibodies against phosphorylated FAK (Tyr397, 3283, Cell Signaling, RRID: AB_2173659 ), phosphorylated ERK 1/2 (S217/221, 9154, Cell Signaling, RRID: AB_2138017 ), P2Y 2 (APR-010, Alomone Labs, RRID: AB_2040078 ), HSC 70 (SC7298, Santa Cruz, RRID: AB_627761 ), or α-tubulin (T5168, Sigma-Aldrich, RRID: AB_477579 ) with 5% BSA in 0.1% TBS-T overnight at 4 °C.

Techniques: Western Blot, CRISPR, Transfection, Labeling

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: Proposed mechanism of P2Y 2 and integrin interactions in pancreatic cancer invasion.

Article Snippet: Following blocking with 5% milk (Sigma) in 0.1% TBS-T for 1 hr, membranes were incubated with 1:1000 dilution of antibodies against phosphorylated FAK (Tyr397, 3283, Cell Signaling, RRID: AB_2173659 ), phosphorylated ERK 1/2 (S217/221, 9154, Cell Signaling, RRID: AB_2138017 ), P2Y 2 (APR-010, Alomone Labs, RRID: AB_2040078 ), HSC 70 (SC7298, Santa Cruz, RRID: AB_627761 ), or α-tubulin (T5168, Sigma-Aldrich, RRID: AB_477579 ) with 5% BSA in 0.1% TBS-T overnight at 4 °C.

Techniques:

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) RNAscope in-situ hybridization of P2Y 2 mRNA expression (magenta) in tumor and matching normal adjacent tissue. ( B ) P2Y 2 mRNA expression in tumor (TCGA) and normal (GTEx) pancreatic tissue samples (*p<0.0001). Graph generated using GEPIA. ( C ) Kaplan-Meier plot comparing patients with high vs low expression of P2Y 2 in the Pancreatic Adenocarcinoma (PAAD) The Cancer Genome Atlas (TCGA) cohort. Graph generated using KMplot. ( D ) Top result of a GSEA (performed with WebGestalt) of two different pancreatic adenocarcinoma patient cohorts (PAAD TCGA and PDAC CPTAC) for the PANTHER pathway functional database. ( E ) Incucyte images of the pancreatic cancer cell line AsPC-1 12 hr after treatment with 100 µM ATP alone or with 5 µM AR-C (P2Y 2 antagonist). Cells are transduced with Lifeact to visualize f-actin (green). ( F ) Schematic of the amino acid sequence of P2Y 2 showing an RGD motif in the first extracellular loop (image generated in http://gpcrdb.org/ ). ( G ) IF staining of P2Y 2 (green), integrin αV (red), and DAPI (blue) in AsPC-1 cells showing colocalization of P2Y 2 and integrin αV (yellow).

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques: RNAscope, In Situ Hybridization, Expressing, Generated, Functional Assay, Transduction, Sequencing, Staining

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) RNAscope in-situ hybridization of a positive control ( PPIB , Cyclophilin B), negative control ( DapB ), and P2Y 2 mRNA expression in a PDAC tissue slide showing tumor and normal adjacent tissue. ( B ) Single-cell expression of P2Y 2 in healthy pancreatic tissue from the Human Protein Atlas ( https://www.proteinatlas.org/ENSG00000175591-P2RY2/single+cell+type/pancreas ). ( C ) Top four results of a GSEA (performed with WebGestalt) of two different pancreatic adenocarcinoma patient cohorts (PAAD TCGA and PDAC CPTAC) for the ‘Molecular Function’ Gene Ontology (GO) functional database. ( D ) Incucyte analysis of average object area related to the average cell area of AsPC-1 cells at different concentrations of ATP (error bars show standard deviation). ( E ) Incucyte images of AsPC-1 cells with different concentrations of AR-C with or without ATP. ( F ) IF staining of four different PDAC cell lines showing various levels of P2Y 2 (green) and integrin αV (red) protein expression. ( G ) The respective reads per kilobase of exon per million reads mapped (RPKM) from CCLE.

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques: RNAscope, In Situ Hybridization, Positive Control, Negative Control, Expressing, Functional Assay, Standard Deviation, Staining

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) Schematic diagram of the hanging drop sphere model for 3D sphere invasion assays. ( B ) Bright field and fluorescent images of spheres formed using AsPC-1 cells (magenta) with a histone 2B (H2B) tagged with a red fluorescent protein (RFP) and the stellate cell line PS-1 (green) with H2B tagged with a green fluorescent protein (GFP). Middle panel shows AsPC-1 cells in spheres with a dotted line highlighting the central sphere area. Spheres were treated with vehicle control or 100 µM ATP alone or with 5 µM AR-C or 10 µM cRGDfV. The quantification is shown in ( C ) using SuperPlots, where each color represents a biological repeat (n=3) and the larger points represent the mean % Invasion for each repeat. ( D ) Quantification of spheres formed by AsPC-1 cells transfected with a control siRNA or P2Y 2 siRNA and treated with or without 100 µM ATP. ( E ) Bright field and fluorescent images of spheres formed by AsPC-1 cells subjected to CRISPR/Cas9 gene disruption using a control guide RNA (CTR CRISPR ) or P2Y 2 guide RNAs (P2Y 2 CRISPR ) and treated with or without 100 µM ATP. Quantification in ( F ). ( G, I ) Bright field and fluorescent images of AsPC-1 P2Y 2 CRISPR cells or PANC-1 cells (respectively) transfected with wild-type P2RY2 (P2Y 2 RGD ) or mutant P2RY2 D97E (P2Y 2 RGE ) treated with or without 100 µM ATP and its quantification in ( H ) and ( J ), respectively. Statistical analysis with Kuskal-Wallis multiple comparison tests.

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques: Control, Transfection, CRISPR, Disruption, Mutagenesis, Comparison

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) Hanging drop sphere with and without PS-1 cells. ( B, C ) Quantification of AsPC-1 spheres treated with 100 µM UTP or ATPγS (respectively) in the absence or together with 5 µM AR-C or 10 µM cRGDfV (n=3 biological replicates). ( D ) IF staining of P2Y 2 in AsPC-1 and PS-1 stellate cells. ( E ) Migration assay with AsPC-1 and 100 µM ATP in the absence or together with 5 µM AR-C or/and 10 µM cRGDfV and ( F ) its quantification (n=3 biological replicates, statistical analysis with one-way ANOVA, * p < 0.5, **** p < 0.0001, error bars show standard deviation). ( G ) 3D sphere invasion assay using BxPC-3 cells treated with 100 µM of ATP in the absence or together with 5 µM AR-C or/and 10 µM cRGDfV and ( H ) its quantification (n=3 biological replicates). ( I ) qPCR of P2Y 2 expression and western blot of siRNA treated cells (control siRNA and P2Y 2 targeting siRNA, n=3 biological replicates). ( J ) AsPC-1 P2Y 2 CRISPR spheres treated with or without 5 µM of AR-C (n=3 biological replicates). ( K ) P2Y 2 IF staining of AsPC-1 P2Y 2 CRISPR cells transfected with an empty vector, P2Y 2 RGD or P2Y 2 RGE plasmids. Statistical analysis of all invasion experiments with Kuskal-Wallis multiple comparison tests. Figure 3—figure supplement 1—source data 1. Labeled uncropped blot of . Figure 3—figure supplement 1—source data 2. Full unedited blot of .

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques: Staining, Migration, Standard Deviation, Invasion Assay, Expressing, Western Blot, Control, CRISPR, Transfection, Plasmid Preparation, Comparison, Labeling

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A, B ) Overview of the DNA-PAINT microscopy technique and qPAINT analysis pipeline. ( C ) Histogram of qPAINT indices for αV (blue) and P2Y 2 (red) single-molecule localization clusters. Solid lines represent multi-peak Gaussian fit. ( D ) Rendered DNA-PAINT images of AsPC-1 P2Y 2 CRISPR cells transfected with P2Y 2 RGD or P2Y 2 RGE with or without 100 µM of ATP and close-ups showing the protein maps reconstructed from DNA-PAINT localization maps of P2Y 2 (red) and integrin αV (cyan). The quantification of the number of proteins or protein clusters (>3 proteins) in each region of interest (ROI) are for P2Y 2 (red) ( E ) and ( F ), respectively and integrin αV (cyan) ( G ) and ( H ), respectively. Quantification of protein proximity using the nearest neighbor distance (NND), with the percentages of integrin αV and P2Y 2 proteins being <50 nm apart ( I ), between different αV integrins being 20–100 nm ( J ) or <20 nm ( K ) apart; and P2Y 2 from other P2Y 2 proteins being <40 nm apart ( H ). Statistical analysis with Kuskal-Wallis multiple comparison test of 21 4x4 µm ROIs from a minimum of 5 cell regions per condition.

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques: Microscopy, CRISPR, Transfection, Comparison

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A ) Schematic diagram of the predicted maximum distance between fluorescent molecules indicating physical contact between proteins, to the nearest first significant figure. ( B ) Histograms of the nearest neighbor distance between proteins vs the frequency of occurrence for AsPC-1 P2Y 2 CRISPR in different conditions (solid line, strong color) or randomly computer-generated controls (dotted line, light color).

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques: CRISPR, Generated

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: ( A, B ) Western blots of phosphorylated FAK (p-FAK) and ERK (p-ERK) of AsPC-1 cells treated with ATP or pre-treated for 30 min with AR-C (5 µM) or cRGDfV (10 µM), respectively and treated with ATP for 60 min. ( C ) Western blot of AsPC-1 P2Y 2 CRISPR cells transfected with P2Y 2 RGD or P2Y 2 RGE and treated with ATP for 60 min. Representative images of three biological replicates. Figure 5—source data 1. Labeled uncropped blots of . Figure 5—source data 2. Full unedited blots of .

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques: Western Blot, CRISPR, Transfection, Labeling

Journal: eLife

Article Title: Purinergic GPCR-integrin interactions drive pancreatic cancer cell invasion

doi: 10.7554/eLife.86971

Figure Lengend Snippet: Proposed mechanism of P2Y 2 and integrin interactions in pancreatic cancer invasion.

Article Snippet: DNA labeling of anti-αV antibody (P2W7, Santa Cruz, RRID: AB_627116 ) and anti-P2Y 2 receptor antibody (APR-010, Alomone labs, RRID: AB_2040078 ) was performed via maleimidePEG2-succinimidyl ester coupling reaction as previously described ( ; ).

Techniques:

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Bar graphs comparing the relative distributions of A 1 R(A1)- and P2Y 2 R(Y2)-immunoreactive elements in each brain region (A-C) and in transfected HEK293T cells (D) . The P2Y 2 R-P2Y 2 R, A 1 R-A 1 R and A 1 R-P2Y 2 R dimers are indicated by Y2-Y2, A1-A1 and A1-Y2, respectively. Total number of immunoreactive gold particles on the cell surface was defined as 100%. Each column represents the average frequency (± SD) from three cells. Raw data are shown in the tables under the graphs. Data are means of three independent experiments.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Transfection

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Co-localization of A 1 R and P2Y 2 R . A-C . Confocal images of double immunostained Myc-P2Y 2 R (A; green), HA-A 1 R (B; red), and their merge (C; yellow) in co-transfected HEK293T cells. The co-localization of HA-A 1 R and Myc-P2Y 2 R is evident at the cell surface membrane (small arrow). D-L . Confocal images of double immunofluorescence staining in several rat brain regions. P2Y 2 R (D, G, J; red) and A 1 R (E, H, K; green) immunoreactivities were detected in Purkinje cells (D-F), cerebellar nuclei (G-I), and hippocampal CA3 pyramidal cells (J-L). Co-localizations of A 1 R and P2Y 2 R (F, I, L; yellow) were detected in the soma (large arrows) of all tissues, in dendrites of the Purkinje cells, and in neurons of the cerebellar nuclei (arrowheads). Yellow bar indicates 500 μm (A-C) and white bar indicates 100 μm (D-L). Mol: cerebellar molecular layer, Gr: cerebellar granule cell layer. Fluorescent images were collected via confocal laser scanning microscopy (Zeiss LSM410, Carl Zeiss, Oberkochen, Germany) each 10-μm optical slice consisted of a stack of 20 0.5-μm thick sections. Serial optical sections were recorded using an air objective lens of (40×, numerical aperture; 0.6).

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Transfection, Double Immunofluorescence Staining, Confocal Laser Scanning Microscopy

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Co-immunoprecipitation of A 1 R from cerebral cortex (lane 1), cerebellum (lane 2), and hippocampus (lane 3) . Immunoblotting analyses of extracts of rat brain with anti-A 1 R (A), anti-P2Y 2 R (B), and anti-P2Y 2 R with the control peptide of P2Y 2 R (C). Membrane extracts from each region were immunoprecipitated with anti-A 1 R, and analyzed by immunoblotting with anti-P2Y 2 R (D), anti-P2Y 2 R with the control peptide of P2Y 2 R (E), anti-A 1 R (F). No bands were seen in C, E demonstrating the specificity of the antibodies. It was confirmed that immunoprecpitation without primary antibody resulted in no detectable receptor bands in the immunoblotting (data not shown). Approximate molecular masses are shown in kDa.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Immunoprecipitation, Western Blot

Journal: BMC Research Notes

Article Title: Immunogold electron microscopic evidence of in situ formation of homo- and heteromeric purinergic adenosine A 1 and P2Y 2 receptors in rat brain

doi: 10.1186/1756-0500-3-323

Figure Lengend Snippet: Immunogold electron microscopy of A 1 R and P2Y 2 R visualized using nanogold particles in transfected HEK293T cells (A-D) and rat brain (E-G) . A: Localization of HA-A 1 R (large particles) detected with anti-HA in HA-A 1 R-transfected HEK293T cells. B: Localization of Myc-P2Y 2 R (small particles) detected with anti-Myc in Myc-P2Y 2 R-transfected HEK293T cells. C: Anti-HA and anti-Myc immuno-localization of HA-A 1 R and Myc-P2Y 2 R in co-transfected HEK293T cells. D: HA-A 1 R-transfected HEK293T cells incubated with both anti-HA and anti-Myc. E-G: Localization of A 1 R and P2Y 2 R in cortical pyramidal cells (E), Purkinje cells (F), and hippocampal pyramidal cells (G) detected with both anti-A 1 R and anti-P2Y 2 R. Arrows indicate two adjacent receptors on the cell membrane. Bars represent 100 nm. CM, cell membrane; CP, cytoplasm.

Article Snippet: Cells were washed and then stained with Alexa 568-conjugated goat anti-rat IgG antibody (1:200, Invitrogen, Carlsbad, CA) for A 1 R or Alexa 488-conjugated goat anti-mouse IgG antibody (1:200, Invitrogen) for P2Y 2 R. The characterization of antibodies for rat brain sections was previously reported, although the rabbit polyclonal anti-P2Y 2 R antibody (anti-P2Y 2 R; 1 μg/ml, Alomone Labs, Jerusalem, Israel) was used instead of the rabbit polyclonal anti-P2Y 1 R antibody [ , ].

Techniques: Electron Microscopy, Transfection, Incubation