Insulin mobilizes both GSVs and GLUT4-containing endosomal compartments. IRAP-pHluorin and TfR-mCherry were cotransfected into 3T3-L1 fibroblast cells (A and C) and adipocytes (B and D), and IRAP-pHluorin fusion events were monitored using dual-color TIRF microscopy 3 min after insulin stimulation for the presence of TfR on the fusing vesicles. (A) An IRAP-pHluorin fusing vesicle with TfR associated with it observed in a 3T3-L1 fibroblast cell. Intensities within the fusion site were measured from both channels and plotted in C. Bar, 0.5 µm. (B) An IRAP-pHluorin fusion event without TfR association observed in an adipocyte. Intensities within the fusion site were measured from both channels and plotted in D. Bar, 0.5 µm. (E) Summary of the presence of TfR on insulin-stimulated IRAP-pHluorin fusing vesicles in 3T3-L1 fibroblasts and adipocytes. Data are represented as mean ± SEM (error bars). Fibroblasts, n = 3 cells and 28 fusions; adipocytes, n = 3 cells and 104 fusions. See also Fig. S2, and Videos 2 and 3.