S6K1 and ribosomal protein S6 are distributed to proplatelets and S6K1 is activated in mature human platelets stimulated with thrombin. (A) Western analysis of total and phosphorylated S6K1 was done using lysates of washed human platelets that were pretreated with rapamycin (10 nM) or vehicle and subsequently stimulated with thrombin as in Figure 1. The bottom panel indicates mobility shift of S6K1 when phosphorylated in response to thrombin activation. The top panel illustrates results of incubations using an antibody that recognizes S6K1 phosphorylated on residues 421 and 424. (B) S6K1 is developmentally expressed in proplatelets and proplatelet tips. Megakaryocytes were cultured from CD34⁺ hematopoietic stem cells as in Figure 1. S6K1 is indicated by red fluorescence and is present in proplatelet tips (arrows). WGA localization is identified by green fluorescence. S6K1 was detected in proplatelet shafts and in proplatelet tips (middle panels, arrows) as proplatelets were extended during developmental transition (also see panel C). In the right panels, nonimmune IgG was substituted for anti-S6K1. (C) Ribosomal protein S6 is distributed to proplatelets and proplatelet tips in megakaryocyte development. S6 protein and WGA are indicated by red and green fluorescence, respectively. CD34⁺ hematopoietic stem cells were differentiated to the megakaryocytic stage.⁴  After 8 days in suspension culture S6 was diffusely distributed in the cytoplasm. One hour after adhesion to immobilized fibrinogen on day 14, S6 was centrally located in the perinuclear cytoplasm. During the transition phase, in which adherent megakaryocytic cells begin to form proplatelets, S6 was diffusely distributed in the central cytoplasm and in early proplatelet shafts. As proplatelets were extended, S6 was detected in early proplatelet tips (bottom right panel, arrows). (B-C) Scale bar, 20 μm.