Immunolocalization of cytoplasmic dynein, dynactin, and kinesin in megakaryocytes. (A) Characterization of antibodies by immunoblotting. Whole-cell protein extracts from mouse megakaryocytes (M), mouse platelets (P), and rat brain (B) were displayed by SDS-PAGE, transferred to PVDF, and immunoblotted with antibodies against β1-tubulin, kinesin, dynein intermediate chain, p50 dynactin, and p150^Glued. Double immunofluorescence microscopy of megakaryocytes using antibodies to (B,D,F) antikinesin and (C,E) either anti-p50 dynactin or (G) anti–dynein intermediate chain antibodies. Immunofluorescence images of preproplatelet megakaryocytes and proplatelet-containing megakaryocytes. (B,F) Kinesin antibodies stain vesicle-like particles within megakaryocytes and (D) along the shafts of proplatelets. (C) p50 dynactin and dynein intermediate chain antibodies diffusely stain the megakaryocyte cytoplasm. Proplatelets are intensely stained (E, inset) along their length with anti-p50 dynactin and (H) anti–dynein intermediate chain antibodies. (H) Comparative immunofluorescent and (I) DIC images of a proplatelet-containing megakaryocyte and mouse platelets (arrows) stained with anti–dynein intermediate chain antibodies. Proplatelets stain robustly with anti–dynein intermediate chain antibody. In contrast, staining of platelets seeded onto the coverslip is highly reduced. Scale bar, 5 μm. Dynactin remains associated with the Triton X-100–insoluble megakaryocyte cytoskeleton (J-M). Megakaryocytes were extracted with 0.5% Triton X-100 in a microtubule-stabilizing buffer before fixation, as described in “Materials and methods.” The cells were then immunostained using (J) kinesin and (L) p50 dynamitin and (K,M) counterstained with α-tubulin antibodies to visualize proplatelet microtubules. Kinesin immunoreactivity was removed by the detergent treatment, suggesting that kinesin is not associated with the microtubules. In contrast, the p50 dynamitin immunoreactivity is preserved.