Figure 3: HGF-driven trafficking of EphA2 is controlled by RCP and Rab14.

(a,b) H1299 cells were transfected with siRNAs targeting RCP (si-RCP) or a non-targeting control (si-Nt) and plated onto 10 cm plastic dishes. Cells were surface-labelled with NHS-S-S-Biotin (0.13 mg ml−1) at 4 °C and internalization allowed to proceed at 37 °C for the indicated times in the presence (a) or absence (b) of primaquine (0.6 mM), with or without HGF (10 ng ml−1). Biotin remaining at the cell surface was removed by exposure to MesNa at 4 °C and the quantity of biotinylated receptors within the cells determined by capture-ELISA using microtitre wells coated with monoclonal antibodies recognizing EphA2. Values are mean±s.e.m. from either one representative experiment performed in quadruplicate (a) or from three independent experiments (b), ***P<0.001 (si-RCP+HGF versus si-Nt+HGF); two-way ANOVA, Bonferroni post-test. (c) H1299 cells expressing GFP-RCP were incubated in the presence or absence of HGF (10 ng ml−1) for 1 min. Cells were lysed in a buffer containing 0.15% Tween-20 and GFP was immunoprecipitated as for Fig. 1c. The immunoprecipitates were analysed by immunoblotting using antibodies recognizing EphA2, Rab14, Rab11 and RCP. Recruitment of EphA2, Rab14 and Rab11 to GFP-RCP immunoprecipitates was quantified by densitometric scanning of western blots. Values are mean±s.e.m. from three independent experiments; *P<0.05, ***P<0.001, ns, not significant; Mann-Whitney test. Uncropped blots corresponding to the experiment presented in Fig. 3c are displayed in Supplementary Fig. 9. (d) H1299 cells were transfected with siRNAs targeting Rab14 (si-Rab14) or a non-targeting control (si-Nt) and internalization of EphA2 in the presence and absence of HGF was determined as for b. Values are mean±s.e.m. from three independent experiments, ***P<0.001 (si-Rab14+HGF versus si-Nt+HGF); two-way ANOVA, Bonferroni post-test. (e,f) H1299 cells were transfected with EphA2-GFP in combination with either mCherry-RCP (e) or mCherry-Rab14 (f) and plated onto glass-bottomed dishes, or were left untransfected. Twenty-four hours following transfection, confocal time-lapse movies were collected with 2 s frame intervals over a 2 min period in the presence and absence of HGF (10 ng ml−1). HGF was added 30 min before collecting the movies. Stills were extracted from movies 3-4 at the indicated time points following collection of the first frame of the movie and display the region of interest indicated by the white boxes. Bar, 10 μm. Olympus software was used to quantify co-localized pixels relative to the EphA2-GFP and mCherry-RCP pixels across the course of the movies, and these are plotted as the fraction of co-localizing pixels as determined by the Costes method39. Values are mean±s.e.m. from three separate experiments incorporating >60 cells per condition, ***P<0.001 Mann-Whitney test. In the right panels in e untransfected H1299 cells were incubated in the presence or absence of HGF (10 ng ml−1) for 30 min and then fixed. Endogenous RCP and EphA2 were visualized by immunofluorescence with respect to the cell nucleus (stained with DAPI) following by high resolution imaging using a high resolution Airy-scan microscope. Detail of the perinuclear region is displayed. Bar, 1 μm. The whole cell fluorescence micrographs and single channel fluorescence images of these Airy-scan images are presented in Supplementary Fig. 4a.