Fig. 8. Overexpressed Hrs inhibits EGF trafficking from early to late endosomes. HEp-2 cells were transfected with myc-tagged Hrs for 6 h and incubated with rhodamine-EGF for 1 h at 37°C (A–F). A portion of the cells was washed and incubated further for 3 h (G–L). The cells were permeabilized with 0.05% saponin prior to fixation. They were stained with anti-myc (A and G) or anti-EEA1 antibodies (C and I) and studied by confocal immunofluorescence microscopy. Turquoise indicates co-localization between Hrs and EEA1 (E and K). Purple indicates co-localization between EGF and EEA1 (F and L). Yellow indicates co-localization between Hrs and EGF (D and J). Bar, 5 µm.