PKCζ and β-catenin are required for polarized Rac1 activation at the migrating front of endothelial cells. (A) Rac1 activity ratio monitored by FRET/cyan fluorescent protein (CFP) time-lapse imaging using Raichu-Rac (YFP-Rac-CFP) at the indicated time points after Ang-1 stimulation of migrating BAECs transfected with CT-siRNA or PKCζ-siRΝΑ. Corresponding yellow fluorescent protein (YFP) fluorescence images are shown in supplemental Figure 4. Neighboring cells are outlined to show the position of Raichu-Rac–transfected cells at the front of migration. Scale bar represents 20 μm. (B) Representative images of corrected FRET after photobleaching for Rac1 activation of a wounded BAEC monolayer expressing the Raichu-Rac probe and transfected with CT-siRNA or PKCζ-siRΝΑ and stimulated or not with Ang-1 (30 minutes). The same cells are shown in fluorescent images of YFP as control for total Rac localization. Neighboring cells are outlined to show the position of Raichu-Rac–transfected cells at the front of migration. Scale bar represents 20 μm. (C) Histogram representing the quantification of Rac activity in fixed BAECs by mean FRET efficiency of the region of interest (leading edge) from all acquisitions from 5 experiments, and data are represented as mean ± SEM (*P < .05 compared with nonstimulated cells). (D) Active Rac-GEF assay showing active Tiam1 in BAEC lysates transfected with CT, PKCζ, or β-catenin-siRNAs. Whole-cell lysates were immunoblotted with anti-PKCζ and β-catenin to confirm down-regulation. Total levels of Tiam1 were used as loading control.