FIG 3.

AAK1 and GAK regulate EGFR-mediated HCV entry. (A and B) Huh-7.5 cells depleted of AAK1 or GAK by siRNAs and NT controls were serum starved and treated with PBS or EGF at the indicated concentrations for 1 h. Cells were subsequently infected with HCVpp (A) or HCVcc (B) expressing a luciferase reporter gene for 4 h, unbound virus was washed off, and luciferase signal was measured at 72 or 24 h postinfection, respectively. Data are relative luciferase values normalized to the corresponding PBS-treated controls (AAK1-depleted, GAK-depleted, or NT) representing fold increase in HCV entry. Data represent means and standard deviations (error bars) from at least three experiments in triplicates. (C to I) Huh-7.5 cells transfected with NT (C and F), AAK1 (D and G), or GAK (E and H) siRNA and treated with either PBS (C to E) or EGF (1 μg/ml) (F to H) were subjected to quantitative confocal IF analysis of EGFR-EEA1 colocalization. Percent colocalization of the indicated signals was measured by a quantitative colocalization analysis of panels C to H using Manders' coefficients (I). Values indicate mean M2 values represented as percent colocalization (the fraction of red intensity that coincides with green intensity) ± standard deviation (error bars; n = 10 to 15). ***, P < 0.001. Representative images at a ×60 magnification of EGFR (red) and the early endosomal marker EEA1 (green) and merged images are shown. Enlargements of insets are shown at the bottom. Scale bar, 10 μm. EE, EEA1.