TUG disruption increases GSV exocytic flux in unstimulated 3T3-L1 adipocytes. (A) Immunoblots of PM fractions from control, TUG shRNA, and rescued (shRNA⁺TUG) cells. The ratio of VAMP2 to TfR abundance is indicated for each sample, normalized to unstimulated control cells. (B) PM VAMP2:TfR was measured in three experiments by fractionation and immunoblotting. Mean ± SEM is plotted (error bars). *, P < 0.05; and ***, P < 0.001 relative to basal control. (C) VAMP2-pHluorin was expressed transiently in control and TUG shRNA cells, and fusion events (crosses) were marked over a 3-min window before (green) and after (red) insulin stimulation (see Videos 3 and 4). Bars, 10 µm. (D) Frequencies of VAMP2-pHluorin and TfR-pHluorin fusion events in control (n = 1,070 from five cells) and TUG shRNA 3T3-L1 adipocytes (n = 895 from five cells). VAMP2-pHluorin events are also plotted for “rescued” TUG shRNA⁺TUG cells (containing shRNA-resistant TUG, n = 262 from three cells). Data are mean ± SEM (error bars). (E) GLUT4-GFP was transfected in control, TUG shRNA, and rescued (TUG shRNA⁺TUG) cells and imaged by TIRFM. Density of single vesicles in the evanescent field of unstimulated cells (n = 20 shRNA, 12 control, and 12 rescued cells; D_p = 98 nm; n = 10; ***, P < 0.001). (F) Percentage of kiss-and-run events in TUG shRNA cells (n = 676 from three cells; **, P < 0.01). Error bars indicate mean ± SEM. (G) Immunoblots were performed as indicated on control and TUG shRNA adipocytes.