Fig. 10. Wortmannin disrupts the association of Hrs and clathrin with transferrin-positive endosomes. HEp-2 cells were incubated with Alexa⁴⁸⁸–transferrin for 30 min at 37°C in the absence (A) or presence (B) of 100 nM wortmannin. The cells were permeabilized with 0.05% saponin prior to fixation in order to remove cytosolic proteins. They were stained with anti-Hrs (left panels) and anti-clathrin (middle panels) and studied by confocal fluorescence microscopy. Arrows indicate examples of co-localization between Hrs, clathrin and internalized transferrin. Arrowheads point at the presumed TGN region stained with anti-clathrin. Bar, 5 µm.