Fig. 4. Colocalisation of Hrs with activated EGFR does not depend on tyrosine phosphorylation of Hrs or on an intact UIM domain. HeLa cells were transfected with GFP-Hrs (A-C), GFP-Y329/334F (D-F) or GFP-UIM (G-I), starved 16 hours in serum-free medium and stimulated 22 hours post-transfection for 8 minutes with EGF (100 ng/ml). The cells were then fixed with 3% PFA, permeabilised with Triton X-100 and stained with anti-EGFR (EGFR shown in red) followed by secondary antibody coupled to Alexa Fluor 594. All panels show a single confocal section of low-expressing cells. Insets show a threefold enlargement of selected areas. Scale bars: 10 µm.