Figure 2: PHD3 interacts with EGFR.

(a) Interaction of endogenous EGFR and PHD3. Proximity ligation assay in G55 cells non-stimulated or stimulated for 5 min with EGF (20 ng ml⁻¹) or exposed to 1% O₂ (hypoxia, 48 h). Red spots are sites of proximity ligation assay amplification reflecting the interaction between EGFR and PHD3, which is enhanced after EGF and hypoxia treatment. (b) Co-immunoprecipitation assay using anti-V5 or anti-EGFR antibodies and extracts from G55 cells expressing PHD3-V5 or GFP control, demonstrating interaction between PHD3 and EGFR. (c) The interaction between EGFR and PHD3 is induced by EGF. Co-immunoprecipitation assay using anti-EGFR antibodies from extracts of G55 cells expressing PHD3-V5 or GFP control and western blot of PHD3 and EGFR. Cells were non-stimulated or stimulated with EGF (20 ng ml⁻¹) for the indicated times. (d) PHD3 co-localizes with EGFR at the plasma membrane and in the early endosomal compartment. Triple co-localization analysis of EGFR, PHD3 and the early endosomal compartment marker EEA1 in G55 cells expressing PHD3 stimulated for 5 min with EGF (20 ng ml⁻¹). Arrows point to sites of triple co-localization. Western blot images (b,c) have been cropped for presentation. Full-size images are presented in Supplementary Fig. 6. All values are means+s.e.m., Student’s t-test ***P<0.001. Scale bars, 10 μm (a), 5 μm (d).