Platelet agonists induce ASK1 phosphorylation. (A) Western blot analysis of lysates from human and murine platelet (free from WBCs) blotted with anti-ASK1 (Cell Signaling; upper panel); reprobed with anti-HSC-70 to ensure equal loading (lower panel). (B-F) Washed human platelets (4 × 108) were stimulated with or without (resting) agonist at different time points, as indicated. Lysates were Western blotted using phosphospecific anti-T845ASK1, which recognizes both human and murine phospho-ASK1. The same blot was reprobed with anti-ASK1 to ensure equal protein loading in all the lanes. Western blot of lysates from resting and thrombin-stimulated platelets (Bi) and the quantitation of band intensity (Bii). Western blot of lysates from resting and ADP stimulated platelets (Ci) and the quantitation of band intensity(Cii). Western blot of lysates from resting and convulxin-stimulated platelets (Di) and the quantitation of band intensity (Dii). Western blot of lysates resting and U46619 stimulated platelet (Ei) and the quantitation of band intensity (Eii). Western blot of lysates from resting and epinephrine-stimulated platelets (Fi) and the quantitation of band intensity (Fii). Band density was calculated by using National Institutes of Health Image J software. All the experiments were repeated more than 3 times using platelets from different individual donors. ns, not significant; Thr, thrombin. ***P < .001.
