Arf6 controls the localization of the Cdc42-GEF βPIX and Cdc42 activation upon wounding. (A) Wound-induced Cdc42 activation in cells nucleofected with the indicated siRNA. (left) Western blots showing a representative experiment. (right) Histogram showing the quantitative measurement of Cdc42 activity. Values are normalized by the Cdc42 activity observed in the control condition (siCTL, time 0). **, P = 0.01. (B) Colocalization of GFP-Cdc42 (green) and HA-βPIX (red) in a migrating astrocyte. (C) Magnified view of the front edge area of the cell shown in B. Arrowheads point to regions showing colocalization of GFP-Cdc42 and HA-βPIX. Fluorescence intensities of GFP-Cdc42 (green) and HA-βPIX (red) along the line drawn in the image show the colocalization of Cdc42 and βPIX at the leading edge and on some of the Cdc42-positive vesicles. Large Cdc42-positive vesicles (asterisks) are usually not stained with the anti-βPIX antibody. (D) βPIX recruitment to the leading edge of astrocytes nucleofected with the indicated siRNA. (left) The percentage of cells with a leading edge accumulation of βPIX. (right) Representative images of βPIX immunofluorescence in cells nucleofected with the indicated siRNA. Higher magnification views of the boxed regions are shown. Dotted lines indicate the direction of the wound. Data are shown as mean ± SEM of three independent experiments totalizing >300 cells. Bars, 10 µm.