A. 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), or eGFP(C-term)-SARA-Fyve domain (PI3P marker), or eGFP(C-term)-PLC-PH domain (PI(4,5) P2 marker), or mkate2(N-term)-2xTAPP1-PH domain (PI(3,4) P2 marker). Representative photomicrographs (from 25 images 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). White bounding boxes show "High Mag" panels that magnify representative individual vesicles. The colocalization of Rab10 and indicated markers in the bounding boxes were analyzed using Image J as described in the method and shown in the "CoLoc" panels. The colocalized pixels are shown in white with the same intensity. Scale bars are 10 μm, or 1 μm in "Hig Mag" panels and "CoLoc" panels. White arrow heads indicate representative Rab10 vesicles positively colocalized with the indicated marker. B. At least 25 cells across three independent experiments were analyzed for each condition (see Materials and Methods section). % of Rab10 vesicles positive with the indicated marker within each cell was quantified. Each dot represents the mean value from one cell analyzed. Group means are given, with error bars representing ± SEM. 