Figure 9.

MTOC reorientation is mediated by Ras-independent pathways. (A) Jurkat T cells were incubated with the indicated concentrations of PD 098059 in DMSO for 30 min, followed by a 10-min stimulation with C305-coated latex beads (+). Untreated control cells were mixed with poly-l-lysine-coated beads (−). Cells were lysed and whole cell lysates were analyzed by immunoblotting with an antiphosphotyrosine antibody. (B) Jurkat T cells were incubated with the indicated concentrations of PD 098059 for 30 min. Subsequently, C305-coated latex beads were added at a 1:2 ratio and conjugates were incubated for 10 min. Conjugates were fixed and stained with antitubulin antibody. The average of four independent experiments is shown. (C) TAg Jurkat cells were cotransfected with TT-εT or TT-ε (30 μg) together with 40 μg of empty vector or a plasmid encoding the dominant-negative Ras mutant N17Ras. 36 h later, cells were mixed with anti–Tac latex beads and incubated for 10 min at 37°C. Conjugates were fixed in paraformaldehyde and stained with antitubulin antibody. The averaged percent MTOC reorientations of two independent experiments are shown. (D) Lysates from 10⁶ Tag Jurkat cells either cotransfected with TT-ε and empty vector or N17Ras were analyzed for the presence of mutant Ras with a Ha-Ras-specific mAb.