Fig. 7. The effects of Akt-PH–GFP mutations on PIP₃ binding, cellular localization and inhibition of cellular responses. (A) COS-7 (upper) and HEK 293 cells (lower) were transfected with the indicated mutant Akt-PH–GFP construct and examined by confocal microscopy without stimulation (HEK 293 cells) or after stimulation with peroxyvanadate (30 µM peroxide, 100 µM ortho-vanadate) for 5-10 minutes (COS-7 cells). (B) Binding of recombinant Akt-PH–GFP mutants to PIP₃ conjugated to agarose beads (sn, unbound fraction in the supernatant; pellet, bound fraction associated with the beads); bars represent the % bound fraction determined by Phosphorimager analysis from three separate experiments (±s.e.m.), one of which is shown as a representative. (C) Inhibition of endogenous Akt activation by Akt-PH–GFP mutants expressed in COS-7 cells. After 24 hours of transfection, cells were stimulated by EGF (100 ng/ml) for 5 minutes. Total cell lysates were analysed by SDS PAGE followed by western blotting using an anti-phospho-Akt antibody and densitometric analysis. In (B) and (C), the R25C mutant, unable to bind the lipid, is indicated by darker columns. (D) Expression kinetics of the various Akt-PH–GFP mutants in COS-7 cells as described in Fig. 6. Fluorescence values were related to those of the non-binding R25C mutant at each time points (mean±s.e.m., n=3). (E) The position of the mutated residue (Thr34) within the Akt-PH domain relative to the PIP₃ binding site in the crystal structure of Akt-PH (1H10).