Colocalization of secreted soluble yellow fluorescent protein (ssYFP) in exocytotic, but not endocytotic, compartments. LLC-AQP2-ssYFP cells were colabeled with fluorescent markers of intracellular compartments (red) and anti-YFP antibodies to highlight all forms [including unfolded or nonfluorescent (green)] of ssYFP. A: extensive colocalization of perinuclear ssYFP and trans-Golgi network marker golgin-97. Extensive colocalization of perinuclear ssYFP and golgin-97 is especially apparent when only native ssYFP fluorescence is visualized (inset). Endocytotic vesicles (B, 15 min of incubation with Alexa 555-dextran), late endosomes/lysosomes (C, 10 min of incubation with LysoTracker Red), and recycling endosomes (D, costaining with anti-Rab11) display no significant colocalization with ssYFP. Rab11 costaining with native ssYFP fluorescence is shown for comparison (D, inset). All colocalization was assessed on 3-dimensional stacks; a single midnuclear xy plane is shown for clarity. Scale bar, 10 μm.