C. Raw 264.7 macrophage cells were transfected with FLAG(N-term)-Rab10 and eGFP(C-term)-Akt-PH domain (PI(3,4,5) P3 marker) and treated with wortmannin (1 μM) for one hour. Representative photomicrographs (from 25 cells analyzed for each condition from n=3 biologically independent experiments) are shown from fixed cells immuno-stained for FLAG-tag (Rab10, shown as green) together with eGFP epifluorescence (shown as red). Scale bars are 10 μm, or 1 μm in "Hig Mag" panel. D. Percent of cells treated with or without wortmannin harboring Rab10 vesicles was calculated from >30 cells across five independent experiments. Each dot on the plot represents mean values from one experiment. Error bars represent ± SEM and significance was assessed by Mann-Whitney test (owing to non-normal distributions) with ** indicating P<0.01. 