Figure 3

FLAG-Eps15 recruitment to GFP-FYVE-UbΔGG-enriched endosomes requires UIM, but not EH or coiled-coil domains. A. Schematic diagram of the Eps15 mutants used. B. FLAG-Eps15 (Eps15) or the indicated FLAG-Eps15 mutant was expressed in COS-7 cells. Proteins in cell lysates (equal volumes) were separated by SDS-PAGE and analyzed by Western blotting, probing with anti-FLAG antibodies. The blot was then probed with anti-Hsp70 antibodies to verify equal loading. C. Wild-type FLAG-Eps15 (Eps15) or the indicated FLAG-Eps1 mutant was expressed in COS-7 cells together with GFP-FYVE-UbΔGG. Cells were processed for IF, detecting the FLAG-Eps15 proteins with anti-FLAG and then AF-594 goat anti-rabbit antibodies, and EEA1 with anti-EEA1 and then AF-647 goat anti-mouse antibodies. From left to right in each row, anti-FLAG staining, anti-EEA1-staining (pseudo-colored blue), GFP fluorescence, and a merged image are shown. Scale bars; 10 μm. D. Wild-type or mutant FLAG-Eps15 was co-expressed with GFP or GFP-FYVE-UbΔGG as indicated, and processed for fluorescence microscopy as in C. Colocalization analysis of the FLAG-Eps15 proteins with EEA1 was performed as in Figure 1. Values shown are the average of 2 experiments, +/- SEM. The data were compared using a one-way ANOVA test (***p < 0.0001, **p < 0.005).