Scribble knockdown disrupts recruitment of Rac1 and Cdc42 to the leading edge. (a) MCF10A cells stimulated with EGF for 10 min and stained for Scribble, βPix and Rac1. (b) Western blot of Rac1-GTP pulldown assay following stimulation of MCF10A cells with EGF (20 ng/ml) for indicated times. GTPγS and GDP loaded controls are shown on the left. Shown below is quantitation of Rac1-GTP levels measured using densitometry averaged from two independent experiments. (c and d) Wounded MCF10A monolayers fixed 6 h after wounding and stained with antibodies to Scribble and Rac1 (c) or Cdc42 (d) and phalloidin to label F-actin. Inset shows a twofold magnification of leading edge cells, identified by a dotted white box.