(A) Thrombopoietin does not induce phosphorylation of pleckstrin or myosin light chain. 32Pi-loaded platelets were stimulated with either thrombopoietin (100 ng/mL) or thrombin (1 U/mL). Whole cell lysates were separated by SDS-PAGE (7.5% to 15% gel). Gels were dried and autoradiographed. Lanes 1 and 6, resting platelets; lanes 2 to 5, 15 seconds, 1 minute, 5 minutes, 10 minutes after addition of thrombopoietin (100 ng/mL); lanes 7 to 9, 15 seconds, 1 minute, 2 minutes after addition of thrombin (1 U/mL). The upper arrow indicates the relative position of pleckstrin; the lower arrow, myosin light chain. (B) Effects of BAPTA-AM and Ro 31-8220 on tyrosine phosphorylation of Vav induced by thrombin or thrombopoietin. Platelets were preincubated with BAPTA-AM (40 μmol/L) and Ro-31-8220 (10 μmol/L) in the presence of 1 mmol/L EGTA for 15 minutes. After the incubation, thrombin (1 U/mL, lane 2) or thrombopoietin (100 ng/mL, lane 3) was added to the platelet suspension. After 10 minutes, platelets were lysed by the addition of an equal amount of a buffer containing 2% Triton X-100. Tyrosine phosphorylation of Vav was detected as in Fig 1 (upper panel). The same PVDF membrane used in the upper panel was stripped and reprobed for Vav (lower panel).
