RacGAP1 is a PKB/Akt substrate required for pseudopod extension and invasion. (A) A2780 cells expressing Akind on CDM were stimulated with 2.5 µM cRGDfV as indicated. Fluorescence lifetime images were captured, and representative lifetime maps are shown. FRET efficiency was calculated for ROI (dotted lines). The yellow line represents the baseline activity as determined by an inactive mutant of the probe. Bar, 10 µm (n > 18/condition). (B) PKB/Akt substrates were immunoprecipitated (IP) using anti-RxRxxS*/T* from lysates of EGF-stimulated cells treated with cRGDfV as indicated. IPs were separated by SDS-PAGE and analyzed by MS/MS. Hierarchical clustering of identified proteins is shown, with increasing abundance indicated by intensity. RacGAP1 is indicated with an asterisk. (C) Purified MBP-RacGAP1 was incubated in in vitro phosphorylation reactions as indicated. Proteins were separated by SDS-PAGE, protein loading was confirmed by Coomassie staining, and incorporation of radioactive ATP was measured by a phosphorimager. (D) Lysates of EGF-stimulated A2780 cells treated with cRGDfV and staurosporine as indicated were subjected to IP using anti-RxRxxS*/T* as in B or an isotype-matched control. IPs were analyzed by SDS-PAGE and Western blotting for RacGAP1 and quantified using the Odyssey system. Data represent means ± SEM from at least three independent experiments. *, P < 0.05; **, P < 0.001.