Rab10 and Rab14 both associate with insulin-stimulated IRAP-pHluorin fusing vesicles. (A–H) Rab4A, Rab8A, Rab10, and Rab14 tagged with TagRFP were separately transfected into adipocytes along with IRAP-pHluorin. IRAP-pHluorin fusion events were monitored using dual-color TIRF microscopy 3 min after insulin stimulation for the presence of a particular Rab protein on the fusing vesicles. (A and E) A Rab4A-negative IRAP-pHluorin fusion event. Fusion site intensities of both channels were measured from A and plotted in E. (B and F) A Rab8A-negative IRAP-pHluorin fusion event. Fusion site intensities of both channels are measured from B and plotted in F. (C and G) A Rab10-positive IRAP-pHluorin fusion event. Fusion site intensities of both channels are measured from C and plotted in G. (D and H) A Rab14-positive IRAP-pHluorin fusion event. Fusion site intensities of both channels are measured from D and plotted in H. Bars, 0.5 µm. (I) Summary of Rab protein associations with insulin-stimulated IRAP-pHluorin fusing vesicles. All Rab proteins were tagged with mKO, and their presence on insulin-stimulated IRAP-pHluorin fusing vesicles was quantified. The association of Rab4A, Rab8A, Rab10, and Rab14 with insulin-stimulated IRAP-pHluorin fusing vesicles was further tested with TagRFP-tagged Rabs. Switching fluorescent protein tags on the Rab proteins had no significant effect on the extent of association. Data are represented as mean ± SEM (error bars). The numbers of cells and insulin-stimulated IRAP-pHluorin fusing vesicles analyzed were as follows: Rab4A, 3 cells and 129 fusions; Rab8A, 3 cells and 136 fusions; Rab10, 3 cells and 143 fusions; and Rab14, 3 cells and 138 fusions. For each of the other Rab proteins, two cells were selected and >60 fusions were examined. The horizontal broken line indicates 20%, which is our threshold for significant association with IRAP-pHluorin fusing vesicles. See also Fig. S3 and Videos 4–7.