Activated Rac3 colocalizes with Scambio to membrane ruffles and binds to residues 1076–1412 of Scambio (A) NIH3T3 cells were co-transfectedwith HA-tagged Scambio and Xpress-tagged V12Rac3. Fibronectin adhered cells were stained for FITC-HA-Scambio (left panel), and Cy3-Xpress-V12Rac3 (middle panel), or merged (right panel). Arrowheads in the right panel point to the numerous ruffles where HA-Scambio and Xpress-V12Rac3 colocalize. Confocal images (× 100), Bar = 20 μm. One cell is shown that is representative of many others obtained in more than two independent experiments. (B) Schematic representation of the clones tested for binding to V12Rac3. Abbreviations used include; HA, hemagglutinin tag; DH, Dbl homology domain; and PH, pleckstrin homology domain. The DH and PH domains are shaded grey and black, respectively. (C) Activated Rac3 binds to a region encompassing the DH-PH domain of Scambio. The DH-PH domain-containing construct including a C-terminal Myc tag was co-transfected with GST-V12Rac3. Binding of this domain to GST-V12Rac3 was assessed on a GST pull-down of the lysate followed by an anti-Myc Western blot.