Fig. 1. EGF-stimulated PtdIns(3,4,5)P₃ production at the leading edge of lamellipod is rapid and sustained. Quiescent MTLn3 cells were stimulated with 5 nM EGF and fixed at various times. (A) Selected images of EGF-stimulated cells stained with anti-PtdIns(3,4,5)P₃ antibody. Bar, 10 µm for all panels. (B) Quantification of leading-edge PtdIns(3,4,5)P₃ production following EGF stimulation. The average edge fluorescence (0-0.66 µm from the perimeter) was measured as described in the Materials and Methods, normalized for the edge fluorescence of unstimulated cells and plotted as a function of time. The data are the mean ± s.e.m. from four experiments. (C) Anti-phosphotyrosine immunoprecipitates from EGF-stimulated cells were assayed for PI 3-kinase activity. The data are the mean ± s.e.m. from four experiments.