Fig. 4. (A–C) Overexpressed Hrs recruits clathrin to early endosomes. BHK cells were transfected with myc-tagged Hrs (A and B) or Hrs1–706 (C) and permeabilized with 0.05% saponin prior to fixation. They were stained with anti-myc (left panels), anti-EEA1 (A, middle panel) or anti-clathrin (B and C, middle panels) and studied by confocal immunofluorescence microscopy. (D–F) Overexpressed Hrs does not recruit AP1, AP2 or AP3. BHK cells or HEp-2 cells (in the case of AP3) were transfected with myc-tagged Hrs and permeabilized with 0.05% saponin prior to fixation. The cells were stained with anti-myc (left panels), anti-AP1 (D, middle panel), anti-AP2 (E, middle panel) or anti-AP3 (F, middle panel) and studied by confocal immunofluorescence microscopy. The right panels show the merged images, with yellow indicating co-localization. Bar, 5 µm.