Fig. 3. Live cell imaging of retrograde STxB sorting on Tf-containing EE/RE. (A) HeLa cells were incubated with Alexa Fluor-488-labeled STxB (green) and Alexa Fluor-568-labeled human Tf (red) for 60 minutes at 19.5°C. After transferring cells to 37°C, they were immediately imaged by time-lapse microscopy. Shown is one cell with STxB and Tf accumulated in early endosomes. Merging images indicate overlapping localization (yellow). Images of the right panels are magnifications of the boxed areas on the left. The series show an STxB-containing tubule moving away from an early endosome (green arrows), while Tf remains associated only with the endosome (red arrows). (B) HeLa cells transiently expressing Vps29-GFP (green) were incubated with Cy3-labeled STxB (red) for 60 minutes at 19.5°C. After transferring cells to 37°C, they were imaged by time-lapse microscopy. Images of the right panels are magnifications of the boxed areas on the left. The series show an STxB-containing tubule moving away from an early endosome (red arrows) and Vps29-GFP associated to the same tubules (green arrows). Time after the start of imaging is shown in seconds. Bars, 10 µm.