anti p2ry12  (Novus Biologicals)

Journal: Blood Advances

Article Title: The Na/K-ATPase α1 subunit fine-tunes platelet P2Y 12 function and mediates sex dimorphism–associated thrombosis

doi: 10.1182/bloodadvances.2025016605

Figure Lengend Snippet: α1 binds to P2Y 12 , and α1 deficiency attenuates ADP-induced AKT activation in platelets. (A) WT platelets pooled from 6 male WT mice were lysed and subjected to co-IP to assess the interaction between NKA α1 and P2Y 12 or P2Y 1 . (B) Platelet lysates from α1 +/− and α1 +/+ mice were analyzed using blue-native polyacrylamide gel electrophoresis (PAGE). The membrane was first probed for α1, then stripped and reprobed for P2Y 12 . The image represents 2 independent experiments. (C) COS-7 cells were transfected with plasmids encoding human P2Y 12 (p-hP2Y 12 ; Addgene number 66471) or the A2B receptor (p-hA2BR; Addgene number 37202) for 36 hours. Cell lysates were then used for co-IP assays to examine α1 binding to P2Y 12 and A2B receptors. (D) PRP from α1 +/− and α1 +/+ mice was pooled from 5 males, adjusted to a final concentration of 2.5 × 10 8 cells per mL using platelet-poor plasma, and divided into 4 aliquots. PRP was supplemented with MgCl 2 /CaCl 2 (1mM final concentration) and stimulated with ADP (2.5μM final concentration) for different time points. The reaction was stopped using an EDTA/PGE1 cocktail, and platelets were lysed for western blot analysis. The blot represents 2 independent experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; IB, immunoblotting; IgG, immunoglobulin G; IP, immunoprecipitation.

Article Snippet: The image represents 2 independent experiments. (C) COS-7 cells were transfected with plasmids encoding human P2Y 12 (p-hP2Y 12 ; Addgene number 66471) or the A2B receptor (p-hA2BR; Addgene number 37202) for 36 hours.

Techniques: Activation Assay, Co-Immunoprecipitation Assay, Polyacrylamide Gel Electrophoresis, Membrane, Transfection, Binding Assay, Concentration Assay, Clinical Proteomics, Western Blot, Immunoprecipitation

Journal: Blood Advances

Article Title: The Na/K-ATPase α1 subunit fine-tunes platelet P2Y 12 function and mediates sex dimorphism–associated thrombosis

doi: 10.1182/bloodadvances.2025016605

Figure Lengend Snippet: LGL mediates the interaction between NKA α1 and P2Y 12 . (A) COS-7 cells were transfected with plasmids encoding mouse WT P2Y 12 along with either WT or LGL→SFT mutant α1. (B-C) COS-7 cells were transfected with plasmids encoding mouse WT α1 along with either WT or LGL→SFT mutant P2Y 12 , and cell lysates were used for co-IP assays (B); α1 band intensity was quantified and expressed as the ratio relative to the average of WT P2Y 12 cotransfected with WT α1 (C). (D) COS-7 cells were transfected with different plasmid combinations and treated with ouabain or digoxin for 36 hours before being lysed for co-IP assays. α1 band intensity was quantified with ImageJ and normalized to lane 1. (E) COS-7 cells were transfected with plasmids encoding human P2Y 12 for 24 hours, then treated with LGL peptide at the indicated dosages for 16 hours. Cells were harvested for co-IP analysis of α1 and P2Y 12 . α1 band intensity was quantified with ImageJ and normalized to the non-LGL condition. GAPDH was blotted as a loading control for input. (F) Washed platelets from male α1 +/− or α1 +/+ mice were resuspended in PBS at 2.5 × 10 8 /mL, and 400 μL was used per aggregation assay. LGL peptide or a leucine-glycine mixture (control) was added to a final concentration of 50μM and incubated for 3 minutes before initiating aggregation. Data were expressed as (LGL – control)/control. An unpaired t test was used. IgG, immunoglobulin G; SB, 2× Laemmli sample buffer.

Article Snippet: The image represents 2 independent experiments. (C) COS-7 cells were transfected with plasmids encoding human P2Y 12 (p-hP2Y 12 ; Addgene number 66471) or the A2B receptor (p-hA2BR; Addgene number 37202) for 36 hours.

Techniques: Transfection, Mutagenesis, Co-Immunoprecipitation Assay, Plasmid Preparation, Control, Concentration Assay, Incubation

Journal: Nature Communications

Article Title: Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice

doi: 10.1038/s41467-026-68885-4

Figure Lengend Snippet: A A mouse model and experimental timeline for targeting microglial Alk5 and analyzing microglia homeostatic marker expression and microgliosis 3 weeks post tamoxifen. B , C Representative images of the hippocampus stained for IBA1, TMEM119, P2RY12 (BioLegend Ab), and GFAP in B Cx3cr1 CreER -ALK5 wt/wt or C Cx3cr1 CreER -ALK5 fl/fl mice D FACS-sorted GFP+ microglia were analyzed for mRNA levels using qRT-PCR showing E Alk5 (indicating gene deletion efficiency), F Tmem119, G P2ry12, H Tnf, and I Igf1 expression levels in control and iKO microglia ( E – I ) ( n = 3 for control, n = 3 for Alk5 KO, * p = 0.0146 for panel E, *** p = 0.0005 for panel F, *** p = 0.0004 for panel G, * p = 0.0372 for panel H, * p = 0.0167 for panel I, two-sided student’s t-test was used for statistical analysis for panels E-I). Representative immunohistochemistry images of IBA1, Ki67, and DAPI in the cortex of J Cx3cr1 CreER -ALK5 wt/wt or K Cx3cr1 CreER -ALK5 fl/fl at 3 weeks after TAM treatment. Arrows show Ki67+ cells (yellow arrows show Ki67+ cells not co-localized with IBA1+ cells and white arrows show Ki67+ cells co-localizing with IBA1+ cells). Unbiased stereological quantification for L Microglia density, M Astrocyte density, N total number of Ki67+ cells, and O the number of IBA1 + /Ki67+ cells ( L – O ) ( n = 4 for control, n = 3 for Alk5 KO, ** p = 0.002, *** p < 0.001, and ** p = 0.001 for panel L, ns = not significant for panel M , ** p < 0.001, p < 0.00, and p = 0.011 for panel N , ** p < 0.001, *** p < 0.001, and ** p = 0.006 for panel O , two-sided 2-way ANOVA with Tukey post hoc pairwise comparison was used for statistical analysis for panels L – O ). Each data point represents the average of a single animal ( > 3 brain sections per mouse for each brain region). The sex of each animal is represented by open circles (females) and open triangles (males). Mean ± SE. Scale bar as indicated. Panel A was created in BioRender. Luo, A. (2026) https://BioRender.com/0xildk2 . Source data are provided as a source data file.

Article Snippet: We generated cell-type-specific inducible knockout models of critical TGF-β signaling components using Cx3cr1 CreER(Jung) (JAX:020940) , Cx3cr1 CreER(Littman) (JAX:021160) , and P2ry12 CreER (JAX:034727) , to delete Alk5 , Tgfbr2 , Igf1 in microglia or Tnfα constitutive knockout.

Techniques: Marker, Expressing, Staining, Quantitative RT-PCR, Control, Immunohistochemistry, Comparison

Journal: Nature Communications

Article Title: Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice

doi: 10.1038/s41467-026-68885-4

Figure Lengend Snippet: A Mouse model for targeting microglial ( P2ry12 CreER/CreER ) Alk5 in adult mice to examine adult neurogenesis in SGZ. B – C Representative images showing immunohistochemistry staining for IBA1, TMEM119, and P2RY12 (Clone P2YM 1E5) in B control mice and C P2ry12 CreER/CreER -Alk5 fl/fl at 3 weeks after TAM administration. D , E Representative images showing immunohistochemistry staining for DCX and Ki67 in D control mice and E P2ry12 CreER/CreER -Alk5 fl/fl at 3 weeks after TAM administration. F – I Quantification for F TMEM119 immunoreactivity, G P2RY12 immunoreactivity, H DCX+ cells quantification compared to the wildtype mean, and I quantification of dendritic arborization (% of immunoreactive positive area) compared to the wildtype mean. F ( n = 6 for WT and n = 6 for KO, **** p < 0.0001); G ( n = 6 for WT and n = 6 for KO, **** p < 0.0001); H ( n = 6 for WT and n = 6 for KO, * p = 0.0359); I ( n = 6 for WT and n = 6 for KO, *** p = 0.0009). Each data point represents the average of a single animal (3–6 brain sections analyzed per mouse) and n = 6 for each group (wildtype or knockout). The sex of each animal is represented by open circles (females) and open triangles (males). Mean ± SE. Two-sided Student’s t-test for all panels except Welch’s t-test was used for panel G. Scale bar as indicated. Panel A was created in BioRender. Luo, A. (2026) https://BioRender.com/1834acf . Source data are provided as a source data file.

Article Snippet: We generated cell-type-specific inducible knockout models of critical TGF-β signaling components using Cx3cr1 CreER(Jung) (JAX:020940) , Cx3cr1 CreER(Littman) (JAX:021160) , and P2ry12 CreER (JAX:034727) , to delete Alk5 , Tgfbr2 , Igf1 in microglia or Tnfα constitutive knockout.

Techniques: Immunohistochemistry, Staining, Control, Knock-Out

Journal: Nature Communications

Article Title: Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice

doi: 10.1038/s41467-026-68885-4

Figure Lengend Snippet: A mouse model used to B micro-dissect the hippocampus and process for single cell 10x Genomic Flex sequencing. C UMAP clustering of cells with annotation for immature neurons and microglia. D Feature plot of Aif1 (coding IBA1 protein) marker to denote microglia cluster. E Feature plot of Dcx to denote DCX+ immature neuroblasts/neuron cluster. F – I Feature plots of Tmem119, P2ry12, Aldh1l1 and Gfap to denote enrichment of microglial signature genes but not astrocytic signature genes in the microglia cluster. J DEGs in MG- Alk5 iKO derived microglia compared to control microglia presented as a volcano plot. K DEGs in MG- Alk5 iKO derived neuroblasts compared to control neuroblasts presented as a volcano plot. L LIANA was used to identify cell-cell interactions and show strong interaction between microglia-endothelial cells and microglia-neuroblasts with identified potential ligand-receptor pairs. M , N EnrichR pathway analysis of DEGs from neuroblasts in MG- Alk5 or Tgfb1 iKO mice with tables denoting top altered disease/cellular processes and molecular pathways. (DEGs) p values were calculated using the Wald test with adjustments for multiple comparisons corrected using the Benjamini-Hochberg method, and (GO Process) p values were calculated using Fisher’s Exact test with adjustments for multiple comparisons were made using Benjamini-Hochberg method. Panel A and B was created in BioRender. Luo, A. (2026) https://BioRender.com/10eqn6o . Source data are provided as a source data file.

Article Snippet: We generated cell-type-specific inducible knockout models of critical TGF-β signaling components using Cx3cr1 CreER(Jung) (JAX:020940) , Cx3cr1 CreER(Littman) (JAX:021160) , and P2ry12 CreER (JAX:034727) , to delete Alk5 , Tgfbr2 , Igf1 in microglia or Tnfα constitutive knockout.

Techniques: Single Cell, Sequencing, Marker, Derivative Assay, Control

Journal: Nature Communications

Article Title: Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice

doi: 10.1038/s41467-026-68885-4

Figure Lengend Snippet: A , B In vivo animal models used and experimental timeline. C Representative images of Cx3cr1 CreER -ALK5 WT/WT or Cx3cr1 CreER -ALK5 fl/fl treated with either Vehicle (VEH) or Rapamycin (RAPA) showing immunohistochemistry staining for DCX and IBA1 3 weeks after Tamoxifen treatment. Yellow Arrowhead shows DCX+ cells migrated out of the SGZ inner layer. D Percentage of total DCX+ cells compared to the wildtype mean and E percentage area of dendritic arborization compared to the wildtype mean. F Percentage of migrated DCX+ cells relative to the total number of DCX+ cells per mouse. G , H Representative images of WT or iKO mice treated with either VEH or RAPA showing immunohistochemistry staining for DCX and pS6 3 weeks after TAM treatment. D – G WT VEH n = 7, KO VEH n = 4, WT RAPA n = 5, and KO RAPA n = 4 (ns=not significant ( D – G ); *** p < 0.001, ** p = 0.01 D ; ** p = 0.003, *** p < 0.001 E ; * p = 0.042, *** p < 0.001 F ; * p = 0.048, ** p = 0.01, *** p < 0.001 G ). The sex of each animal is represented by open circles (females) and open triangles (males). I In vitro experimental model and timeline for primary adult NSCs and microglia ( P2ry12 CreER/CreER - tdTomato) 3D co-culture. J Representative images of a differentiated neuroimmune 3D coculture sphere showing microglial tdTomato, GFAP and TUJI immunostaining (maximum projection from Z stacked confocal images). K , L Representative images of the 3D coculture spheres quantification of the TUJI immunoreactive density in the spheres after differentiation with indicated microglia genotypes and VEH or RAPA treatment (WT VEH n = 4, iKO VEH n = 6, WT RAPA n = 6, iKO RAPA n = 4) (ns=not significant, * p = 0.0352, *** p = 0.001 K ). M A proposed model of mechanistic action through microglia onto adult-born neurons. Mean ± SE. Scale bar = 100 µm, Two-way ANOVA test, two-sided, with Tukey post hoc pairwise comparison was used for statistical analysis for all panels. Panel A , B , I and M were created in BioRender. Luo, A. (2026) https://BioRender.com/sxk2w6x . Source data are provided as a source data file.

Article Snippet: We generated cell-type-specific inducible knockout models of critical TGF-β signaling components using Cx3cr1 CreER(Jung) (JAX:020940) , Cx3cr1 CreER(Littman) (JAX:021160) , and P2ry12 CreER (JAX:034727) , to delete Alk5 , Tgfbr2 , Igf1 in microglia or Tnfα constitutive knockout.

Techniques: In Vivo, Immunohistochemistry, Staining, In Vitro, Co-Culture Assay, Immunostaining, Comparison

Journal: Nature Communications

Article Title: Inhibition of TGF-β signaling in microglia stimulates hippocampal adult neurogenesis and reduces anxiety-like behavior in adult mice

doi: 10.1038/s41467-026-68885-4

Figure Lengend Snippet: A The mouse model for single Alk5 iKO or double Alk5/Igf1 iKO and experimental timeline (right) for targeting microglial Igf1 and/or Alk5 and analyzing immature neurons 3 weeks post tamoxifen. Representative images of immunohistochemistry staining of the hippocampus from tamoxifen-treated (3 weeks post) A Cx3Cr1 CreER(WT/WT) , B Cx3Cr1 CreER -Igf1 fl/fl , C Cx3Cr1 CreER -Alk5 fl/fl , D Cx3CR1 CreERT2 -Alk5 fl/fl /Igf1 fl/fl tissue showing IBA1, TMEM119, P2RY12 (P2YM 1E5) staining. E – G Representative DCX staining in SGZ in above mouse lines. Quantification of mRNA levels from Cx3Cr1 CreER(WT/WT) , Cx3Cr1 CreER -Alk5 fl/fl , or Cx3CR1 CreERT2 -Alk5 fl/fl /Igf1 fl/fl sorted microglia (normalized to Hmbs1) showing levels of H Hprt1 , I Iba1 , J Alk5 , and K Igf1 relative to Cx3Cr1 CreER(WT/WT) mice( n = 3 for all groups in panels ( H – K )) (ns=not significant ( H – I ); **** p < 0.0001 and ns=not significant J ; *** p = 0.0001 and p = 0.0001 and ns=not significant K ). L Quantification of the number of DCX+ cells and M dendritic arborization of DCX+ cells compared to WT mean ( n = 12 for WT, 4 for Alk5 KO, and 7 for Igf1 / Alk5 KO L ; n = 10 for WT, 4 for Alk5 KO, and 7 for Igf1 / Alk5 KO M ) (** p = 0.0071 and p = 0.0011 and ns=not significant for panel L ; **** p < 0.0001 *** p = 0.0005 and ns=not significant M ). The sex of each animal is represented by open circles (females) and open triangles (males). Mean ± SE. One-way ANOVA, two-sided, with Tukey post hoc pairwise comparison was used for statistical analysis in all panels ( H –M). Scale bar = 100 µm. Panel A was created in BioRender. Luo, A. (2026) https://BioRender.com/egkiitm . Source data are provided as a source data file.

Article Snippet: We generated cell-type-specific inducible knockout models of critical TGF-β signaling components using Cx3cr1 CreER(Jung) (JAX:020940) , Cx3cr1 CreER(Littman) (JAX:021160) , and P2ry12 CreER (JAX:034727) , to delete Alk5 , Tgfbr2 , Igf1 in microglia or Tnfα constitutive knockout.

Techniques: Immunohistochemistry, Staining, Comparison

Journal: bioRxiv

Article Title: Microglia and its P2RY12 Receptors Regulate Seizure Severity

doi: 10.64898/2025.12.11.693689

Figure Lengend Snippet: a, Time course of modified Racine seizure scores in P2RY12-deficient mice. b , Quantification of peak seizure score in P2RY12 ⁻/⁻ mice (p < 0.05, unpaired t-test). c, Kaplan–Meier survival analysis of survival probability in P2RY12 ⁻/⁻ mice (p < 0.05, log-rank test). N = 11-18 mice each. d , Representative confocal images of IBA1 (green) and P2RY12 (magenta) immunostaining in cWT(P2RY12 fl/fl :CX3CR1 Cre/+ ) and conditional knockout cKO (P2RY12 fl/fl :CX3CR1 Cre/+) mice confirm selective microglial deletion of P2RY12. e , Quantification of P2RY12 mean fluorescence intensity in cWT and cKO mice (unpaired t-test). N = 3 mice each. f , Tail bleeding assay comparing platelet function across genotypes confirming microglia-specific targeting (p < 0.0001, one-way ANOVA with Tukey’s post hoc test). N = 4-8 mice each. g-i , Modified Racine seizure scores overtime of cWT and cKO ( g ), peak seizure score ( h , p < 0.01), and Kaplan–Meier survival analysis ( i ). N = 10-13 mice each.

Article Snippet: This study used both male and female mice on a C57BL/6J background between 2-4 months of age, and consisted of the following genotypes: C57Bl/6J mice as wildtype mice; CX3CR1 GFP/+ expressing GFP under control of the fractalkine receptor (CX3CR1) promoter (Jackson Lab, #005582); P2RY12 KO mice; CX3CR1 Cre mice (Jackson Lab, #025524); P2RY12 fl/fl mice as a generous gift from Dr. Long-Jun Wu at the University of Texas Health in Houston; Csf1r ΔFIRE/ΔFIRE as a generous gift from Dr. Sandro da Mesquita, Mayo Clinic.

Techniques: Modification, Immunostaining, Knock-Out, Fluorescence

Journal: bioRxiv

Article Title: Microglia and its P2RY12 Receptors Regulate Seizure Severity

doi: 10.64898/2025.12.11.693689

Figure Lengend Snippet: a-d , Representative in vivo two photon images of microglia from P2RY12 WT, HET and KO littermates (a) used to quantify process protraction (b), retraction (c) and velocity (d). N = 3 mice each. Data shown as mean ± SEM. Statistics assessed by an ANOVA.

Article Snippet: This study used both male and female mice on a C57BL/6J background between 2-4 months of age, and consisted of the following genotypes: C57Bl/6J mice as wildtype mice; CX3CR1 GFP/+ expressing GFP under control of the fractalkine receptor (CX3CR1) promoter (Jackson Lab, #005582); P2RY12 KO mice; CX3CR1 Cre mice (Jackson Lab, #025524); P2RY12 fl/fl mice as a generous gift from Dr. Long-Jun Wu at the University of Texas Health in Houston; Csf1r ΔFIRE/ΔFIRE as a generous gift from Dr. Sandro da Mesquita, Mayo Clinic.

Techniques: In Vivo

Journal: bioRxiv

Article Title: Microglia and its P2RY12 Receptors Regulate Seizure Severity

doi: 10.64898/2025.12.11.693689

Figure Lengend Snippet: a–d , Representative confocal images ( a, c ) of IBA1 + microglia and quantification of the ramification index ( b, d ) from hippocampal CA1 ( a-b ) and the somatosensory cortex ( c-d ) regions in wildtype (WT) and P2RY12 ⁻/⁻ (KO) littermate mice under baseline (0 min) and post-seizure (90 min) conditions following kainic acid administration. Data shown as mean ± SEM. N = 4-6 mice each. Statistics done by two-way ANOVA with Tukey’s post hoc test.

Article Snippet: This study used both male and female mice on a C57BL/6J background between 2-4 months of age, and consisted of the following genotypes: C57Bl/6J mice as wildtype mice; CX3CR1 GFP/+ expressing GFP under control of the fractalkine receptor (CX3CR1) promoter (Jackson Lab, #005582); P2RY12 KO mice; CX3CR1 Cre mice (Jackson Lab, #025524); P2RY12 fl/fl mice as a generous gift from Dr. Long-Jun Wu at the University of Texas Health in Houston; Csf1r ΔFIRE/ΔFIRE as a generous gift from Dr. Sandro da Mesquita, Mayo Clinic.

Techniques:

Journal: bioRxiv

Article Title: Microglia and its P2RY12 Receptors Regulate Seizure Severity

doi: 10.64898/2025.12.11.693689

Figure Lengend Snippet: a–b , Representative confocal images of cFos immunoreactivity from the hippocampal CA1 ( a ) and somatosensory cortex ( b ) regions in wildtype (WT) and P2RY12 ⁻/⁻ (KO) mice under baseline (0 min) and post-seizure (90 min) conditions following kainic acid administration. c , Quantification of mean cFos fluorescent intensity in the CA1 (left) and cortex (right) across genotypes and time points. N = 4 mice each. Data shown as mean ± SEM. Statistics by two-way ANOVA with Tukey’s post hoc test.

Article Snippet: This study used both male and female mice on a C57BL/6J background between 2-4 months of age, and consisted of the following genotypes: C57Bl/6J mice as wildtype mice; CX3CR1 GFP/+ expressing GFP under control of the fractalkine receptor (CX3CR1) promoter (Jackson Lab, #005582); P2RY12 KO mice; CX3CR1 Cre mice (Jackson Lab, #025524); P2RY12 fl/fl mice as a generous gift from Dr. Long-Jun Wu at the University of Texas Health in Houston; Csf1r ΔFIRE/ΔFIRE as a generous gift from Dr. Sandro da Mesquita, Mayo Clinic.

Techniques:

Journal: bioRxiv

Article Title: Microglia and its P2RY12 Receptors Regulate Seizure Severity

doi: 10.64898/2025.12.11.693689

Figure Lengend Snippet: a–b, Representative confocal images of VGAT immunoreactivity from the hippocampal CA1 ( a ) and somatosensory cortex ( b ) regions in wildtype (WT) and P2RY12 ⁻/⁻ (KO) mice under baseline (0 min) and post-seizure (90 min) conditions following kainic acid administration. c , Quantification of VGAT-labeled area per μm of tissue section in the CA1 (left) and cortex (right) across genotypes and time points. N = 4 mice each. Data shown as mean ± SEM. Statistics by two-way ANOVA with Tukey’s post hoc test.

Article Snippet: This study used both male and female mice on a C57BL/6J background between 2-4 months of age, and consisted of the following genotypes: C57Bl/6J mice as wildtype mice; CX3CR1 GFP/+ expressing GFP under control of the fractalkine receptor (CX3CR1) promoter (Jackson Lab, #005582); P2RY12 KO mice; CX3CR1 Cre mice (Jackson Lab, #025524); P2RY12 fl/fl mice as a generous gift from Dr. Long-Jun Wu at the University of Texas Health in Houston; Csf1r ΔFIRE/ΔFIRE as a generous gift from Dr. Sandro da Mesquita, Mayo Clinic.

Techniques: Labeling