Ask1 regulates TxA₂generation through activation of p38 MAPK. (A-B) Western blots of lysates of WT and Ask1^−/− platelets stimulated with thrombin (Thr), ADP, or collagen and blotted using phosphospecific anti-ERK1/2 (Ai) or phosphospecific anti-JNK1/2 (Bi). The same blots were reprobed with anti-ERK1/2 or anti-JNK1/2 to ensure equal protein loading in all the lanes. (Aii, Bii) Quantitation of data. (C) Western blots of lysates of WT and Ask1^−/− platelets stimulated with AYPGKF for various time points and blotted using phosphospecific anti-p38. The same blot was reprobed with anti-p38 to ensure equal protein loading in all the lanes. Shown is a representative blot from 3 independent experiments. (D) Western blots of lysates from panel A blotted using phosphospecific anti-p38. The same blot was reprobed with anti-p38 to ensure equal protein loading in all the lanes. (E-G) Western blots of lysates of WT and Ask1^−/− platelets stimulated with thrombin for indicated time points and blotted using phosphospecific anti-MKK3/6 (E), phosphospecific anti-MKK4 (F), or phosphospecific anti-MKK7 (Gi). The same blot was reprobed with anti-MKK3 (E), anti-MKK4 (F), or anti-MKK7 (Gi) to ensure equal protein loading in all the lanes. Shown is a representative blot from 3 independent experiments. (Gii) Quantitation of band density from panel Gi. (H) Quantitation of band density of Western blot of lysates from panel A, blotted using phosphospecific anti-MKK7 normalized to total MKK7. ns, not significant. *P< .05; **P< .01.