FIGURE 1:

(A) INS-1 cells were transfected with NPY-emdGFP-SBP together with the TGN marker sialyltransferase-TagRFP657 and ER-hook, incubated with biotin for the indicated times, fixed, and imaged using a widefield epifluorescence microscope. (B) INS-1 cells transfected with RUSH constructs were incubated with biotin for 1 or 24 h, fixed, and immunostained for insulin followed by Alexa Fluor 647–conjugated secondary antibodies. Cells were imaged by spinning-disk confocal microscopy. Insets denoted by a square are shown in B’. INS-1 (C, D) or PC12 cells (E) were transfected with NPY-emdGFP-SBP and ER hook for secretion assays. Cells were incubated with biotin for 1.5 h (C, E) or 3 h (D) followed by exposure to basal or stimulated conditions for 15 min. Secretion was determined by reading the fluorescence in the media and was normalized to total fluorescence in cell lysate. *p < 0.05, ****p < 0.0001 relative to NPY basal (for INS-1 cells 1.5 h, n = 3, for INS-1 cells 3 h, n = 4, for PC12 cells n = 3) by unpaired two-tailed t test. Data shown indicate mean ± SEM. Scale bar indicates 10 μm and 1 μm for insets.