p2x3  (Alomone Labs)

Journal: Purinergic Signalling

Article Title: Expression of the P2X1 receptor remains in the type II spiral ganglion neurons in the mature rat cochlea

doi: 10.1007/s11302-026-10129-7

Figure Lengend Snippet: P2X1 receptor expression in the adult Wistar rat cochlea. Cryosections ( A - B , E ) and whole mount ( C - D , F ) from adult (6–8 week-old) Wistar rat cochleae were immunolabelled with anti-βIII tubulin ( green ) and anti-P2X1 antibody ( red ), and counterstained with DAPI ( blue ). (A-B) Representative image showing the overviews of rat cochlear cryosections ( A ) and the region with the organ of Corti and Rosenthal’s canal ( B ) showing P2X1 receptor expression in the organ of Corti (OoC) and the spiral ganglion neurons (SGN). (C-D) 6–8-week-old rat cochlea whole mount OoC preparation showing P2X1 immunolabelling, focusing on the SGN processes underneath the inner and outer hair cells ( C ) and the corresponding region in P2X1 antibody peptide block control ( D ). ( E -E”) Rosenthal’s canal of cryosection from ( B ). ( F -F’) 6–8-week-old OoC rat cochlea whole mount z-stack images showing tunnel crossing fibres labelled with anti-P2X1 antibody (arrowheads) and P2X1 immunolabelling at the base of the OoC (*). Scale bars, 50 μ m (A), 40 μ m ( B - E ), 20 μ m ( F ). Representative images from n = 4 cochleae from four different animals

Article Snippet: The primary antibodies used were: rabbit polyclonal anti-P2X1 antibody (Alomone Labs, Jerusalem, Israel, Catalog# APR-001, 1:500 dilution), mouse anti-β III tubulin (IgG2a monoclonal, BioLegend, San Diego, CA, catalog No. 801213, 1:1000 dilution), goat polyclonal anti-peripherin-1 antibody (Everest Biotech, Upper Heyford, UK, catalog No. EB12405, 1:1000 dilution).

Techniques: Expressing, Blocking Assay, Control

Journal: Purinergic Signalling

Article Title: Expression of the P2X1 receptor remains in the type II spiral ganglion neurons in the mature rat cochlea

doi: 10.1007/s11302-026-10129-7

Figure Lengend Snippet: Co-expression of P2X1 receptors and peripherin-1 in Wistar rat cochlea. ( A - C ) Cryosections from adult (6–8 week-old) Wistar rat cochleae were immunolabelled with anti-βIII tubulin (green) and anti-P2X1 antibody (red), and counterstained with DAPI (blue). ( F ) White arrows indicate SGNs with strong P2X1 signals relative to other SGNs. Images were taken of the basal ( A ), mid-( B ) and apical ( C ) regions of the cochlea. Scale bar = 20 μm for ( A - C ). ( D - G ) Cryosections from 6–8-week-old Wistar rat cochlea ( D - E ) and P8 cochlea ( F - G ) were co-labelled with anti-peripherin-1 antibody ( green ), and anti-P2X1 antibody ( red ). Images show the overview from the apical turn ( F ) and spiral ganglion neurons in the apical region ( G -G”). Representative images from n = 4 cochleae from four different animals

Article Snippet: The primary antibodies used were: rabbit polyclonal anti-P2X1 antibody (Alomone Labs, Jerusalem, Israel, Catalog# APR-001, 1:500 dilution), mouse anti-β III tubulin (IgG2a monoclonal, BioLegend, San Diego, CA, catalog No. 801213, 1:1000 dilution), goat polyclonal anti-peripherin-1 antibody (Everest Biotech, Upper Heyford, UK, catalog No. EB12405, 1:1000 dilution).

Techniques: Expressing

Journal: Science Advances

Article Title: A genome-wide genetic screen reveals the P2Y2-integrin axis as a stabilizer of EGFR mutants in non–small cell lung cancer (NSCLC)

doi: 10.1126/sciadv.adv3980

Figure Lengend Snippet: ( A ) Micrographs of EGFR-19del-GFP stable cells transfected with siRNAs targeting control (siControl), EGFR (siEGFR), or P2Y2 (siP2Y2) from two independent screens. ( B ) Quantification of EGFR-19del-GFP intensity in individual cells from both screens. ( C ) Immunohistochemistry staining of P2Y2 in tumor specimens from 29 patients with NSCLC, including EGFR-WT and EGFR-mutant cases. Representative immunohistochemistry images shown; scale bar, 75 μm. ( D ) P2Y2 protein expression quantified using the H -score and analyzed by a one-way ANOVA. ( E ) P2Y2 mRNA levels (normalized to actin) measured in NSCLC cell lines, grouped by EGFR status (WT: blue; mutant: red). Statistical analysis via a one-way ANOVA with Tukey’s test ( P = 0.0006). H1299, A549, H1299-19del, PC9, H1975, and H1650 cells were transfected with siControl or siP2Y2. Knockdown efficiency was confirmed by quantitative PCR ( F ), and EGFR, phospho-AKT, phospho-ERK1/2, and actin levels were assessed by immunoblotting ( G ). EGFR mRNA levels were also quantified ( H ). ( I ) H1299-EGFR-19del cells were transfected with siControl or siP2Y2, serum starved, and treated with BafA1 (500 nM) or MG132 (10 μM) for 6 hours. EGFR and actin levels were assessed by immunoblotting. ( J ) EGFR-19del-GFP internalization following EGF stimulation in siControl or siP2Y2 cells. Cells were fixed at indicated time points and stained for Lamp1 and DAPI; images are from a confocal microscope. Matrigel invasion ( K ) and cell proliferation ( M ) assays were performed in NSCLC cells transfected with siControl or siP2Y2. Invaded area quantified using ImageJ ( L ); proliferation measured over 96 hours using CyQUANT assay (M). Statistical analysis: one-way ANOVA with Tukey’s post hoc test. * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001. Scale bar, 10 μm. a.u., arbitrary units. OD, optical density. ns represents nonsignificant differences.

Article Snippet: Endogenous peroxidase was quenched with 3% hydrogen peroxide for 10 min, followed by blocking with 3% blocking buffer for 5 min. An anti-P2Y2 antibody (Alomone Labs, APR-010) and anti-integrin β1 (D2E5) antibody (CST, no. 9699) were used at 1:3000 and 1:100, respectively.

Techniques: Transfection, Control, Immunohistochemistry, Staining, Mutagenesis, Expressing, Knockdown, Real-time Polymerase Chain Reaction, Western Blot, Microscopy, CyQUANT Assay

Journal: Science Advances

Article Title: A genome-wide genetic screen reveals the P2Y2-integrin axis as a stabilizer of EGFR mutants in non–small cell lung cancer (NSCLC)

doi: 10.1126/sciadv.adv3980

Figure Lengend Snippet: ( A ) Immunoblotting analysis on H1975 cells, with control and with P2Y2 knocked out via CRISPR-Cas9 (P2Y2-KO1 and P2Y2-KO2). EGFR and its downstream signaling events (pAKT and pERK) along with total AKT and total ERK were analyzed. Actin serves as the loading control. Quantification of ( B ) apoptotic cells using TUNEL (terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling) assay and ( C ) colony-forming ability using clonogenic assay was performed on H1975 cells and P2Y2 KO cells. ( D ) Xenograft tumor growth assay was performed on H1975 and P2Y2-KO1 cells by subcutaneously injecting 3 × 10 6 cells, and ( E ) monitoring of the tumor volume for up to 46 days was carried out to analyze their tumorigenicity in vivo. The tumor volume of H1975 compared to H1975 P2Y2-KO1 shows a statistically significant difference using an ANOVA test, with P < 0.0001. ( F ) Immunoblot analysis of the EGFR and actin levels of EGFR-WT and EGFR mutants (19del, 19del-T790M, and 19del-T790M-C797S) upon siRNA-based knockdown of P2Y2 (siP2Y2) or control (siControl) in H1299 stable cells (left). Analysis of mRNA expression of P2Y2, EGFR, and actin using RT-PCR (right). ( G ) Quantification of the EGFR protein levels normalized to actin in three independent experiments. Statistical analyses were performed using a ANOVA, and Tukey post hoc test was selected. * P ≤ 0.05, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: Endogenous peroxidase was quenched with 3% hydrogen peroxide for 10 min, followed by blocking with 3% blocking buffer for 5 min. An anti-P2Y2 antibody (Alomone Labs, APR-010) and anti-integrin β1 (D2E5) antibody (CST, no. 9699) were used at 1:3000 and 1:100, respectively.

Techniques: Western Blot, Control, CRISPR, TUNEL Assay, End Labeling, Clonogenic Assay, Growth Assay, In Vivo, Knockdown, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: Science Advances

Article Title: A genome-wide genetic screen reveals the P2Y2-integrin axis as a stabilizer of EGFR mutants in non–small cell lung cancer (NSCLC)

doi: 10.1126/sciadv.adv3980

Figure Lengend Snippet: ( A ) Immunoblot of the expression levels of the iATPSnFR1.0 probe (using an anti-GFP antibody), EGFR, and actin in HEK293 cells stably expressing EGFR-WT and EGFR mutants (19del, 19del-T790M, and 19del-T790M-C797S) using a GFP-based single-wavelength ATP sensor transfected with cell surface ATP probe—iATPSnFR1.0. ( B ) Micrographs of GFP fluorescence representing extracellular ATP levels in HEK stable cells with bright-field images in the bottom panel. The bar graph represents quantification of the GFP intensity of the ATP sensor. Immunoblots using EGFR and actin antibodies on H1975 ( C ) and H1299 ( D ) cells that were treated with ATP (100 μM). Graphs on the bottom are the quantification of EGFR protein from the immunoblot normalized to actin. hrs, hours. ( E ) Immunoblots of H1975 and H1299 cells treated with apyrase (10 U/ml) and then harvested at the specified time points. The bottom panels represent the EGFR and actin mRNA expression analysis using RT-PCR. Graphs on the bottom are the quantification of EGFR protein from the immunoblot normalized to actin. ( F ) siRNA-based knockdown of P2Y2 (siP2Y2) or control (siControl) in H1975 cells treated with or without ATP (100 μM). Cells were harvested after 1 hour and immunoblotted for EGFR or actin (left), quantification of the EGFR protein levels normalized to actin in three independent experiments (middle) was performed, and mRNA expression of EGFR and actin using RT-PCR (right) was determined. Actin serves as the loading control. Statistical analyses were performed using an ANOVA, and Tukey post hoc test was selected; * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Endogenous peroxidase was quenched with 3% hydrogen peroxide for 10 min, followed by blocking with 3% blocking buffer for 5 min. An anti-P2Y2 antibody (Alomone Labs, APR-010) and anti-integrin β1 (D2E5) antibody (CST, no. 9699) were used at 1:3000 and 1:100, respectively.

Techniques: Western Blot, Expressing, Stable Transfection, Transfection, Fluorescence, Reverse Transcription Polymerase Chain Reaction, Knockdown, Control

Journal: Science Advances

Article Title: A genome-wide genetic screen reveals the P2Y2-integrin axis as a stabilizer of EGFR mutants in non–small cell lung cancer (NSCLC)

doi: 10.1126/sciadv.adv3980

Figure Lengend Snippet: ( A ) Schematic showing P2Y2 embedded in the plasma membrane, highlighting the RGD motif in green. Created in BioRender. G. T. K. Boopathy (2025); https://biorender.com/yklytxg . ( B ) Dot plot of normalized EGFR-19del intensity scores for 27 integrin genes from two replicates of the genome-wide RNAi screen. Blue, red, and green dots represent siControl, siEGFR, and siP2Y2, respectively; black dots indicate individual integrin genes, with selected gene names labeled. ( C ) Micrographs of EGFR-19del-GFP cells treated with siRNAs targeting β1 ( ITGB1 ), β4 ( ITGB4 ), β5 ( ITGB5 ), and EGFR (siEGFR) from the RNAi screen. H1975 cells treated with ( D ) ATP (100 μM) or ( E ) apyrase (10 U/ml) and harvested at indicated time points. Lysates were immunoblotted for phospho-Y397-FAK (pFAK), total FAK, and actin. ( F ) H1975 cells pretreated with or without the RGD peptide and stimulated with or without ATP for 30 min or 1 hour. Lysates were immunoblotted for EGFR, pFAK, total FAK, and actin. ( G ) H1975 cells treated with an integrin β1–blocking antibody (AIIB2) at indicated concentrations for 16 hours. Lysates were immunoblotted for EGFR, pFAK, total FAK, and actin. ( H ) EGFR, integrin β1, and actin levels in H1299 EGFR-WT, H1299 EGFR-19del, H1299, and H1975 cells following integrin β1 knockdown (siβ1) or siControl. After 72 hours, cells were serum starved for 16 hours and lysed. EGFR band intensities were normalized to actin and indicated on the blot. ( I ) Representative immunohistochemistry images of integrin β1 (ITGB1) in NSCLC tissues from 29 patients (13 EGFR-WT and 16 EGFR-mutant). ( J ) Quantification of ITGB1 protein expression ( H -score) from (I). Statistical analysis: one-way ANOVA; * P < 0.05. Scale bar, 75 μm.

Article Snippet: Endogenous peroxidase was quenched with 3% hydrogen peroxide for 10 min, followed by blocking with 3% blocking buffer for 5 min. An anti-P2Y2 antibody (Alomone Labs, APR-010) and anti-integrin β1 (D2E5) antibody (CST, no. 9699) were used at 1:3000 and 1:100, respectively.

Techniques: Clinical Proteomics, Membrane, Genome Wide, Labeling, Blocking Assay, Knockdown, Immunohistochemistry, Mutagenesis, Expressing

Journal: Science Advances

Article Title: A genome-wide genetic screen reveals the P2Y2-integrin axis as a stabilizer of EGFR mutants in non–small cell lung cancer (NSCLC)

doi: 10.1126/sciadv.adv3980

Figure Lengend Snippet: Coimmunoprecipitation using ( A ) an anti-IgG or anti-EGFR antibody and ( B ) an anti-P2Y2 (anti–FLAG-M-agarose) antibody in H1975 cell lysates transfected with P2Y2-FLAG and immunoblotted with anti-integrin β1, anti-FLAG, and anti-EGFR antibodies. Coimmunoprecipitation using an anti-IgG or anti-EGFR antibody on ( C ) H1299 cell lysates treated with and without ATP (100 μM; 5 min) and ( D ) H1975 cell lysates with ATP (100 μM; 1 hour) with or without apyrase (10 U/ml; 1 hour) and immunoblotted with anti-integrin β1, anti-FLAG, anti-EGFR, and actin antibodies. Band intensities of EGFR, integrin β1, and P2Y2 were quantified and normalized to immunoprecipitated EGFR, with normalized values indicated below the respective blots. ( E ) EGF-induced internalization of EGFR-WT-GFP or EGFR-19del-GFP, tdTomato-integrin β1, and P2Y2 (FLAG immunofluorescence) was detected at the indicated time points in the presence of ATP and demonstrates the colocalization of complex members during EGFR signaling. DAPI stains the nucleus. ( F ) Coimmunoprecipitation of EGFR-19del-GFP with P2Y2 in H1299 cells treated with either DMSO or the RGD (50 μM) peptide for 16 hours. The cell lysates and immunoprecipitates were blotted for GFP (EGFR), FLAG (P2Y2), pFAK, FAK, and actin. ( G ) Coimmunoprecipitation of EGFR-19del-GFP with P2Y2-FLAG in H1299 cells treated with DMSO or kaempferol (50 μM) for 16 hours. The cell lysates and immunoprecipitates were blotted for GFP (EGFR), FLAG (P2Y2), and actin. Scale bar, 10 μm.

Article Snippet: Endogenous peroxidase was quenched with 3% hydrogen peroxide for 10 min, followed by blocking with 3% blocking buffer for 5 min. An anti-P2Y2 antibody (Alomone Labs, APR-010) and anti-integrin β1 (D2E5) antibody (CST, no. 9699) were used at 1:3000 and 1:100, respectively.

Techniques: Transfection, Immunoprecipitation, Immunofluorescence

Journal: Science Advances

Article Title: A genome-wide genetic screen reveals the P2Y2-integrin axis as a stabilizer of EGFR mutants in non–small cell lung cancer (NSCLC)

doi: 10.1126/sciadv.adv3980

Figure Lengend Snippet: HEK-EGFR-19del-T790M-C797S stable cells were treated with the indicated doses of ( A ) third-generation EGFR TKI (osimertinib), ( B ) P2Y2 inhibitor (kaempferol), and ( C ) FAK inhibitor (PF-562271) and analyzed for cell proliferation using the CyQUANT assay for 4 days. The heatmap graph illustrates the percentile scores of EGFR-19del-T790M-C797S cell proliferation using combination therapies: ( D ) kaempferol with osimertinib, ( E ) PF-562271 with osimertinib, and ( F ) kaempferol with PF-562271, presented as average fluorescence intensities. ( G ) Schematic model of the stability of EGFR-WT and mutants. The left panel depicts ATP levels and EGFR degradation in normal cells, the middle panel represents EGFR-mutant tumor cells, and the right panel outlines strategies to target EGFR stability (represented in red). Ab, antibody. Created in BioRender. G. T. K. Boopathy (2025); https://biorender.com/yklytxg . ( H ) Schematic representation of the stability of EGFR mutants resulting from the increased extracellular ATP levels, activating P2Y2 in complex with integrins and stabilizing EGFR. Created in BioRender. G. T. K. Boopathy (2025); https://biorender.com/yklytxg . ( I ) Proposed model for comprehensive targeting of diverse EGFR mutants in NSCLC using a P2Y2 inhibitor, NTPDase, FAK inhibitor, or a combination with TKIs to overcome TKI resistance. Created in BioRender. G. T. K. Boopathy (2025); https://biorender.com/yklytxg . Statistical analysis was performed using an ANOVA. ** P ≤ 0.01, *** P ≤ 0.001, and **** P ≤ 0.0001.

Article Snippet: Endogenous peroxidase was quenched with 3% hydrogen peroxide for 10 min, followed by blocking with 3% blocking buffer for 5 min. An anti-P2Y2 antibody (Alomone Labs, APR-010) and anti-integrin β1 (D2E5) antibody (CST, no. 9699) were used at 1:3000 and 1:100, respectively.

Techniques: CyQUANT Assay, Fluorescence, Mutagenesis