Figure 4.

ASK1 regulates the phosphorylation of P2Y₁₂ at Thr345 in platelets. (a) P2Y₁₂ expression in platelets of Wt, Ask1^−/−, Ask1^F/F and Pf4-cre; Ask1^F/F mice (n=2 each). (b) Platelet lysates of Wt and Ask1^−/− mice were subjected to buffer or λPPase to examine the impact of dephosphorylation on the bands for P2Y₁₂. (c) Intracellular consensus phosphorylation sites for JNK and p38 ((S/T)P sites) in mouse P2Y₁₂ were compared with those of human P2Y₁₂. The SP site of mouse P2Y₁₂ denoted in blue is not conserved in human P2Y₁₂. (d) Evaluation of the specificity of the phospho-P2Y₁₂ Thr³⁴⁵ antibody. CHO cells exogenously expressing mP2Y₁₂-WT, T345A or control lacZ were stimulated with 10 μM ADP for 5 min, lysed and subjected to dephosphorylation by λPPase. (e) Western blot of mouse platelets of Wt, Ask1^−/−, Ask1^F/F and Pf4-cre; Ask1^F/F mice (n=2 each) (arrowhead; P-P2Y₁₂ Thr³⁴⁵ or P2Y₁₂, *nonspecific band)