Figure 2.

(A–D) Insulin regulates subcellular localization of dFOXO. S2 cells overexpressing dFOXO or dFOXOA3 were grown in the absence (A,C) or presence (B,D) of insulin. (E) Akt phosphorylates and inhibits dFOXO activity. (Top) S2 cells grown in the absence of serum/insulin were transfected with dFOXO [wild-type (WT) lanes 1,4], dFOXOA3 (A3, lanes 2,5), or with empty vector (C, lanes 3,6). Myr-dAkt-V5 was cotransfected in lanes 4–6. (Bottom) Luciferase assays performed with the samples from above and measured with a reporter containing four FOXO4 recognition elements. (F) Insulin inhibits dFOXO through dAkt. Cells were transfected with dFOXO (lanes 2,4) or dFOXOA3 (lanes 1,3) and dsRNA against dAkt (lanes 1,2) or lacI (lanes 3,4) was added. (Bottom) Luciferase activity was measured with the same reporter as in E.