Figure 2.

GFP-2xFYVE does not inhibit bulk transport to late endosomes in vivo. (A) As in Fig. 1 A, a pulse of rhodamine dextran was endocytosed with or without chase in cells expressing a double FYVE domain associated to GFP (GFP-2xFYVE). Cells were analyzed as described in the legend to Fig. 1 A. (B) Cells expressing or not expressing GFP-2xFYVE were incubated overnight with 40 μg/ml mouse IgGs with or without 50 μg/ml leupeptin. Endocytosed IgGs were revealed using anti-mouse antibodies and cells were analyzed by double channel fluorescence microscopy. The total number of labeled vesicles (containing IgGs in red) was counted for each condition in control cells or cells expressing GFP-2xFYVE. Vesicles in ≥20 cells were counted for each condition in three different experiments. Numbers were similar whether GFP-2xFYVE was present or not, and are expressed as a percentage of the total number of vesicles in leupeptin-treated cells, to facilitate direct comparison. Bar, 2.5 μm.