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