Fig. 5. EGFR downregulation is inhibited by over-expression of Hrs independently of tyrosine phosphorylation. HeLa cells were transfected with GFP-Hrs (A-C), GFP-Y329/334F (D-F) or GFP-UIM (G-I), starved for 16 hours in serum-free medium and treated 22 hrs post-transfection for 4 hours 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 Fuor 594. All panels show a single confocal section of cells presenting the most typical staining pattern for each construct. Scale bars: 20 µm.

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