Figure 6.

Membrane-targeted PKB/Akt induces GLUT4 externalization in both control and nelfinavir-treated 3T3-L1 adipocytes. 3T3-L1 adipocytes were electroporated in the presence of plasmids encoding a membrane-targeted PKB/Akt (myr Akt) and Myc-GLUT4-GFP. Cells were then treated or not with 30 μm nelfinavir (Nelf) for 18 h. A, After rinsing the cells, they were stimulated for 10 min with 100 nm insulin, after which cells were fixed, permeabilized, and stained with anti-pS473-PKB/Akt or anti-pT308-PKB/Akt. B, Cells were left unstimulated with insulin, fixed, and stained for surface myc. C and D, Rims of myc staining or the myc to GFP fluorescence intensity ratio, respectively, were rated as described in Materials and Methods, and results of 20–30 cells in each of eight independent experiments are shown.