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. 2005 Oct 7;102(42):15081–15086. doi: 10.1073/pnas.0502889102

Fig. 1.

Fig. 1.

Development of InPAkt. (a) A cartoon depicting the conformational change upon PI binding, yielding a FRET response. (b) Domain structure of the construct showing the restriction sites linking individual components. (c) Anti-GFP Western blot of InPAkt expressed in HEK 293 cells indicating the right size of the reporter. (d) FRET response of InPAkt expressed in NIH 3T3 cells. Yellow fluorescence images show membrane translocation of InPAkt upon PDGF stimulation. Pseudocolor images indicate the emission ratio change at various time points after PDGF stimulation. (e) Representative emission ratio time courses of InPAkt (n = 9) and the PH domain mutant R23A/R25A (n = 2), both stimulated with 50 ng/ml PDGF. InPAkt showed a response of 25.4 ± 3.7% (average ± SD; n = 9). (f) A representative emission ratio time course from two independent experiments showing that the response of InPAkt is PI3K-specific. NIH 3T3 cells were pretreated with 20 μM PI3K inhibitor LY294002, followed by stimulation with 50 ng/ml PDGF, gentle washing, and treatment with PDGF again. (g) A representative emission ratio time course shows the reversibility of the reporter. NIH 3T3 cells expressing InPAkt were stimulated with PDGF, followed by treatment with 20 μM LY294002 (n = 5).