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Fig. 3. Dephosphorylation of cofilin does not occur at the leading edge following stimulation with EGF. (A) MTLn3 cells were fixed at 0 and 60 seconds following stimulation with EGF and stained with antibodies against cofilin and p-cofilin. The F-actin cytoskeleton was stained with Alexa Fluor-633-labeled phalloidin. The dephosphorylated cofilin image was prepared by subtracting the scaled p-cofilin image from the total cofilin image using Image J software. (Bar, 10 µm.) (B) Immunofluorescence quantification of dephosphorylated cofilin and p-cofilin throughout the whole cytoplasm of resting and stimulated cells 60 seconds after stimulation with EGF. (Error bars indicate the s.e.m.) (C) Fluorescence intensity analysis of dephosphorylated cofilin and p-cofilin throughout the lamellipod. The lamellipod is defined as the region extending from the cell edge to 2.5 µm into the cell interior, whereas the leading edge is the region where most of the actin nucleation occurs and extends 1 µm from the membrane (Chan et al., 2000; DesMarais et al., 2002). (Error bars indicate the s.e.m.)

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