human p2y 12  (Addgene inc)

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: 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: PLoS ONE

Article Title: Characterisation of P2Y 12 Receptor Responsiveness to Cysteinyl Leukotrienes

doi: 10.1371/journal.pone.0058305

Figure Lengend Snippet: HEK293 cells were transiently transfected with indicated vectors and intracellular responses recorded. (A) [h-CysLT 1 ] and (B) [h-CysLT 2 ] transfectants stimulated with indicated concentrations of LTC 4 , LTD 4 and LTE 4 . N = 6. (C) Flow cytometry analysis of h-P2Y 12 protein surface expression in [h-P2Y 12 ] and [Empty] transfectants using an anti-HA antibody, representative of three experiments. (D) [h-P2Y 12 ] and [Empty] transfectants stimulated with ADP or LTE 4 , N = 9. Results (A), (B) and (D) represented as peak intracellular calcium response, relative fluorescence units (RFU), mean ± S.E.M.

Article Snippet: HEK293 cells cultured to above 60% confluence were transiently transfected with a mixture of Lipofectamine 2000 (Life Technologies) and the following plasmids as indicated: pcDNA3.1-human CysLT 1 , pcDNA3.1-human CysLT 2 , pcDNA3.1-human P2Y 12 , pcDNA3.1-3xHA human P2Y 12 , pcDNA3.1-human Gα 16 , pcDNA3.1-3xHA human ADRB2 (all the Missouri S&T cDNA Resource Center, Rolla, Mo) and pCMV6-Kan/Neo- mouse P2Y 12 (Origene Technologies) in serum-free medium (Opti-MEM, Life Technologies) according to manufacturer’s protocol.

Techniques: Transfection, Flow Cytometry, Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Characterisation of P2Y 12 Receptor Responsiveness to Cysteinyl Leukotrienes

doi: 10.1371/journal.pone.0058305

Figure Lengend Snippet: HEK293 cells were transiently transfected with vectors indicated and intracellular responses recorded. (A) Flow cytometry analysis of ADRβ 2 and h-P2Y 12 protein surface expression in [ADRβ 2 + G α16 ] and [hP2Y 12 + G α16 ] transfectants respectively, representative of three experiments. ADRβ 2 and h-P2Y 12 were detected using an anti-HA antibody, representative of three experiments. (B) [ADRβ 2 ] and [ADRβ 2 + G α16 ] transfectants stimulated with isoproterenol, N = 6, 2-way ANOVA p = 0.0003. [h-P2Y 12 + G α16 ] and [Empty+G α16 ] transfectants stimulated with LTE 4 and either (C) 2-MeS-ADP or (D) ADP, N = 9. 2-way ANOVA between (C) 2-MeS-ADP responses p = 0.0190 and (D) ADP responses p = 0.0220. Results (B), (C) and (D) presented as peak intracellular calcium response mean ± S.E.M.

Article Snippet: HEK293 cells cultured to above 60% confluence were transiently transfected with a mixture of Lipofectamine 2000 (Life Technologies) and the following plasmids as indicated: pcDNA3.1-human CysLT 1 , pcDNA3.1-human CysLT 2 , pcDNA3.1-human P2Y 12 , pcDNA3.1-3xHA human P2Y 12 , pcDNA3.1-human Gα 16 , pcDNA3.1-3xHA human ADRB2 (all the Missouri S&T cDNA Resource Center, Rolla, Mo) and pCMV6-Kan/Neo- mouse P2Y 12 (Origene Technologies) in serum-free medium (Opti-MEM, Life Technologies) according to manufacturer’s protocol.

Techniques: Transfection, Flow Cytometry, Expressing

Journal: PLoS ONE

Article Title: Characterisation of P2Y 12 Receptor Responsiveness to Cysteinyl Leukotrienes

doi: 10.1371/journal.pone.0058305

Figure Lengend Snippet: Intracellular cAMP concentrations and β-arrestin recruitment was analysed in models of transiently transfected HEK293 or stably modified CHO cells, respectively. (A) [h-P2Y 12 ] and [Empty] transfectants were stimulated with forskolin and either ADP or LTE 4 , N = 6, data expressed as % of forskolin stimulated control. (B) CHO cells expressing h-P2Y 12 and β-arrestin were stimulated with either 2-MeS-ADP or LTE 4 , N = 9, expressed as relative luminescence units (RLU). Data represented as mean ± S.E.M. Two-way ANOVA with Bonferroni post-hoc test, *p<0.05.

Article Snippet: HEK293 cells cultured to above 60% confluence were transiently transfected with a mixture of Lipofectamine 2000 (Life Technologies) and the following plasmids as indicated: pcDNA3.1-human CysLT 1 , pcDNA3.1-human CysLT 2 , pcDNA3.1-human P2Y 12 , pcDNA3.1-3xHA human P2Y 12 , pcDNA3.1-human Gα 16 , pcDNA3.1-3xHA human ADRB2 (all the Missouri S&T cDNA Resource Center, Rolla, Mo) and pCMV6-Kan/Neo- mouse P2Y 12 (Origene Technologies) in serum-free medium (Opti-MEM, Life Technologies) according to manufacturer’s protocol.

Techniques: Transfection, Stable Transfection, Modification, Expressing

Journal: PLoS ONE

Article Title: Characterisation of P2Y 12 Receptor Responsiveness to Cysteinyl Leukotrienes

doi: 10.1371/journal.pone.0058305

Figure Lengend Snippet: HEK293 cells were transiently transfected with indicated vectors and intracellular calcium responses recorded. (A) [m-P2Y 12 + G α16 ] and [Empty+G α16 ] transfectants were stimulated with LTE 4 and 2-MeS-ADP, N = 9, 2-way ANOVA between 2-MeS-ADP responses p = 0.0101. (B) CHO cells stably expressing m-P2Y 12 and β-arrestin were stimulated with either 2-MeS-ADP or LTE 4 , N = 9. Data presented as mean ± S.E.M.

Article Snippet: HEK293 cells cultured to above 60% confluence were transiently transfected with a mixture of Lipofectamine 2000 (Life Technologies) and the following plasmids as indicated: pcDNA3.1-human CysLT 1 , pcDNA3.1-human CysLT 2 , pcDNA3.1-human P2Y 12 , pcDNA3.1-3xHA human P2Y 12 , pcDNA3.1-human Gα 16 , pcDNA3.1-3xHA human ADRB2 (all the Missouri S&T cDNA Resource Center, Rolla, Mo) and pCMV6-Kan/Neo- mouse P2Y 12 (Origene Technologies) in serum-free medium (Opti-MEM, Life Technologies) according to manufacturer’s protocol.

Techniques: Transfection, Stable Transfection, Expressing

Journal: Scientific Reports

Article Title: Ticagrelor Enhances Release of Anti-Hypoxic Cardiac Progenitor Cell-Derived Exosomes Through Increasing Cell Proliferation In Vitro

doi: 10.1038/s41598-020-59225-7

Figure Lengend Snippet: SNS314 mesylate (SNS314 M), an inhibitor phosphorylation of histone H3 at serine 10 (phospho-H3S10), reduces cell viability ( A ) and proliferation ( B ) in a dose dependent manner in hCPCs untreated and treated with ticagrelor (Tic 1 μM). Higher dose of SNS314 M (10 μM) counteracts ticagrelor (Tic 1 μM) effects ( A , B ). SNS314 M 10 μM counteract effects of Tic 1 μM on levels of phospho-H3S10/H3 ratio ( C ) without altering expression of P2Y 12 ( D ; 52KDa MW) of hCPCs. Levels of phospho-H3S10, H3 and P2Y 12 are normalized on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) levels and expressed as arbitrary units (a.u.). Representative images of cropped densitometric bands of proteins phospho-H3S10, H3, P2Y 12 and GAPDH are showed in panel E. The full-length blots/gels are presented in Supplemental Fig. panel A. All measurements are mean ± SD. *p < 0.05 vs. untreated condition (Vehicle: sterile phosphate buffer solution); # p < 0.05 vs. Tic 1 μM; § p < 0.05 vs. SNS314 M (10 μM).

Article Snippet: Primary antibodies were used to detect: (1) human CD63 (polyclonal antibody, 1:1000; #SAB4301607, Sigma-Aldrich Chemical Co, MO, USA) and human TGS101 (polyclonal antibody, 1:1000; #T5701, Sigma-Aldrich Chemical Co, MO, USA) which are established exosomal markers ; (2) human heat shock protein 70 (HSP70) (polyclonal antibody, 1:1000; #ADI-SPA-812F, Enzo Life Sciences, NYC, USA), an antiapoptotic protein even used as exosomal marker ; (3) human P2Y 12 on surface of CPCs (polyclonal antibody, 1:1000; #SC27152, Santa Cruz Biotechnology Inc., USA); (4) human phosphorylated Histone H3 on Serine 10 (phospho-H3S10) (polyclonal antibody, 1:1000; #9701 S, Cell Signaling, Danvers, MA) and human histone H3 (H3) (monoclonal antibody, 1:1000; #4499 S, Cell Signaling, Danvers, MA), which ratio is an established hallmark of cell proliferation ; (5) murine cleaved caspase-3 (polyclonal antibody, 1:1000; #SC7148, Santa Cruz Biotechnology Inc., USA), a marker of cell apoptosis ; (6) murine phospho-ERK42/44 (polyclonal antibody, 1:1000; #SC23759, Santa Cruz Biotechnology Inc., USA) and murine ERK42/44 (polyclonal antibody, 1:2000; #SC94, Santa Cruz Biotechnology Inc., USA) in HL-1 cells, which ratio is an established hallmark of exosome-mediated pro-survival pathway , ; (7) murine hypoxia inducible factor 1-alpha (HIF1α; monoclonal antibody, 1:1000; #ab179483, Abcam, UK), a transcription factor activated by low oxygen concentrations ; (8) glyceraldehyde 3-phosphate dehydrogenase (GAPDH; monoclonal antibody, 1:3000; #G8795, Sigma-Aldrich Chemical Co, MO, USA) as loading control for protein normalization.

Techniques: Expressing