Figure 5: PHD3 regulates the recruitment of Eps15 and epsin1 to EGFR.

(a,b) PHD3 loss increases Grb2, Cbl-b and c-Cbl recruitment to EGFR, as well as EGFR ubiquitination. Immunoblot of Grb2 and Cbl-b (a) and of c-Cbl and ubiquitin (b) of immunoprecipitated EGFR from G55 cells expressing control or PHD3 shRNA that were non-stimulated or EGF stimulated for the indicated times. (c) Recruitment of the endocytic adaptor Eps15 is impaired following PHD3 loss. Immunoblot of Eps15 of immunoprecipitated EGFR from G55 cells expressing control or PHD3 shRNA that were non-stimulated or EGF stimulated for the indicated times. (d,e) PHD3 and Eps15 interact. Co-immunoprecipitation assay using anti-V5 antibodies or IgG control from extracts of G55 cells expressing PHD3-V5 and Eps15-Flag (d) or PHD3-V5 (e) and western blot of Eps15 and PHD3, demonstrating interaction of PHD3 with exogenous and endogenous Eps15, respectively. Cells were non-stimulated or stimulated with EGF (20 ng ml⁻¹) for 5 min. (f) Recruitment of the endocytic adaptor epsin1 is impaired following PHD3 loss. Immunoblot of epsin1 of immunoprecipitated EGFR from G55 cells expressing control or PHD3 shRNA that were non-stimulated or EGF stimulated for 2 min. Western blot images (a–f) have been cropped for presentation. Full-size images are presented in Supplementary Fig. 7.