Figure 3.

HIV-1 infection promotes membrane recruitment of Akt's PH domain, resulting in increased Akt activity. (A) Primary human macrophages were sequentially infected with M-tropic HIV-1 YU-2 and Ad.CMV-EGFP-PHAkt expressing the PH domain of Akt, and localization of the PH domain of Akt was assessed by fluorescence microscopy. Heat inactivated YU-2 was used as a negative control, and treatment with epidermal growth factor (EGF) was used as a positive control for Akt activation. HIV-1 infected macrophages were treated with 10 μM Miltefosine (Milt.) for inhibition of Akt. BF: bright field. GFP: green fluorescent protein. Inset: High magnification images of representative cells. The percentage of membrane localized PH-Akt is shown with the SD from three independent experiments. (B) Assay for Akt kinase activity. Macrophage and CHME5 cells were transduced with HIV vector and lysed. Using these lysates, an Akt kinase activity assay was performed using GSK3β as a substrate. Western blots of phospho-GSK3β (GSK3β-P) and α-Tubulin (loading control) are shown along with the fold induction of Akt kinase activity relative to control. Fold increase of Akt kinase activity is also shown. The error bars denote the SD from three independent experiments.