Multiple Rab proteins reside on GLUT4 vesicles. (A) Rab proteins tagged with EGFP and GLUT4-mCherry were cotransfected into adipocytes, and their colocalization was examined in the absence of insulin stimulation using dual-color TIRF microscopy. Bars, 4 µm. (B) Quantification of Rab protein colocalization with GLUT4 vesicles in the absence of insulin stimulation. All Rab proteins were tagged with mKO and quantified for their colocalization with GLUT4-EGFP vesicles. Rab4A, Rab4B, Rab8A, Rab10, and Rab14, which showed overlap with GLUT4-EGFP vesicles, were then tagged with EGFP and further tested with GLUT4-mCherry. Switching fluorescent protein tags in this manner had no significant effect on the extent of colocalization. v/n, Rab vesicles were observed close to the PM but had no colocalization with GLUT4; n, no Rab vesicle were observed close to the PM; #, Rab proteins that showed frequent docking behavior after insulin stimulation. Data are represented as mean ± SEM (error bars). The number of cells and GLUT4 vesicles analyzed is as follows: Rab4A, n = 3 cells and 144 vesicles; Rab4B, n = 3 cells and 137 vesicles; Rab8A, n = 3 cells and 153 vesicles; Rab10, n = 3 cells and 190 vesicles; and Rab14, n = 3 cells and 140 vesicles. See also Table S1 and Fig. S1. (C) mKO-tagged Rab proteins were transfected into adipocytes, and 3 min after insulin stimulation their movement beneath the PM was compared with insulin-responsive GLUT4-EGFP vesicle docking processes using TIRF microscopy. Bars, 0.64 µm.