Fig. 1. Cdc42 activation at the protruding edge in response to EGF stimulation. MTLn3 cells were serum-starved for 3 hours and then stimulated with 5 nM EGF at 37°C for the indicated times. (A) GST-CRIB pull-downs of unstimulated and stimulated cells. Anti-Cdc42 western blots showing activated (GTP-Cdc42) and total Cdc42. The data are representative of three experiments. (B) GST-CRIB assay for Cdc42 activation in cells pre-treated with carrier (DMSO) or 100 nM wortmannin (Wm) for 15 minutes, then stimulated with EGF. (C) Representative micrographs of EGF-stimulated cells, fixed at various times and immunostained with anti-Cdc42 antibody. (D) Quantification of data shown in C; fluorescence intensity in the lamellipod edge (0.2-0.6 µm in from the edge of the cell) was normalized to edge intensity of non-stimulated cells. The data is the mean ±s.e.m. from three different experiments (15 cells per experiment), plotted as a function of time. (E) MTLn3 cells were starved for 3 hours and microinjected with the Cdc42 biosensor as explained in Materials and Methods. After 1 hour, cells were stimulated with 5 nM EGF for various times, or not (0 minutes), and fixed. Ratio images (I-SO:GFP) reflect the activation (GTP binding) of Cdc42 at different times after EGF stimulation. Activation was maximal at the 180 seconds, with regions of highest activity five times greater than in the unstimulated cells. (F) At each time point, the mean ratio from a region of interest (2 µm inwards from the edge to the center of the cell) is calculated as fold increase relative to unstimulated cells, and plotted as a function of time. Data are the mean ± s.e.m. from 40 different cells. Bars, 10 µm.