Fig. 5. Reversible translocation of PI 3-kinase to the plasma membrane and phosphorylation of Akt upon addition of excess phospholipids. (A) Superimposed optical sections (focused on the middle of the cell body) of PI 3-kinase (green) and F-actin (red) recorded by confocal microscopy. (B,C) Immunoblotting of phosphorylated Akt of total platelets lysates treated in different conditions. (B) (lane 1) Control; (lanes 2 and 3) in the presence of excess PC at 1 and 5 minutes, respectively; (lanes 4 and 5) the same conditions as lane 2 but in the presence of 100 nM wortmannin and 50 µM LY294002, respectively; (lanes 6 and 7) in the presence of excess PS at 1 and 30 minutes, respectively; (lane 8) the same conditions as lane 6 but in the presence of 100 nM wortmannin; (lane 9) platelets activated by 1 U/ml of thrombin. (C) Platelets in the presence of excess PC after a 1 minute (lane 1) and 30 minute (lane 2) incubation. The data are representative of three individual experiments. Bar, 1.5 µm (A).