S6K1 and Rac1 localize to the lamellipodial edge of spreading platelets. (A) Purified human platelets were spread on coverglass coated with 25 μg/mL fibrinogen. After 45 minutes, platelets were fixed, stained for S6K1, Rac1, and actin, and visualized by confocal microscopy. Scale bar = 2 μm. (B) S6K1 was immunoprecipitated (IP) from lysates of quiescent platelets in solution or platelets spread on fibrinogen and analyzed for coprecipitating Rac1 by Western blot. Nonspecific rabbit immunoglobulins (IgG) were used as negative control for immunoprecipitations. Total S6K1 and Rac1 levels in whole-platelet lysates serve as input controls. (C) Platelet lysates were incubated with GST or Rac1-GST glutathione sepharose and captured S6K1 was analyzed by Western blot. Coomassie-stained GST and Rac1-GST inputs are shown. (D) TIAM1 was immunoprecipitated from platelet lysates as above and examined for coprecipitating S6K1 and Rac1 by Western blot. (E) Localization of TIAM1, Rac1 and actin in fibrinogen-activated human platelets visualized by confocal microscopy. Scale bar = 2 μm. Western blot, IP, protein capture, and imaging results are representative of 3 independent experiments.