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Figure 2


Fig. 2. The kinetics of EGF-stimulated Ras and Rac activation and translocation. (A,B) GST-CRIB (from hPAK) or GST-RBD (from Raf 1) were immobilized on glutathione-Sepharose beads and incubated with EGF-stimulated cell lysates as described. Glutathione-Sepharose pull-downs were separated by 14% SDS-PAGE gel and blotted with anti-Rac or anti-Ras antibody. (A, upper panel) A representative anti-Rac immunoblot of a GST-CRIB pull-down from EGF-stimulated cells. (Lower panel) A representative anti-Ras immunoblot of a GST-RBD pull-down from EGF-stimulated cells. Where indicated, cells were treated with 100 nM wortmannin prior to EGF stimulation. Total Ras and Rac levels did not change during 5 minutes of EGF stimulation (data not shown). (B) Ras (closed diamonds) and Rac (open circles) pull-down assays were quantified by densitometry. Data were expressed as percentage of maximum activity, and show the mean ± s.e.m. from nine experiments. (C) Representative fluorescent images of MTLn3 cells stimulated with EGF for various times and stained with anti-Rac antibodies. Bar, 10 µm. (D) Representative images of EGF-stimulated MTLn3 cells stained with GST-RBD followed by anti-GST antibody, to visualize endogenous activated Ras. Bar, 10 µm.





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