Fig. 6. Ena/VASP proteins are necessary for the internalisation of p-Eph in response to cell surface-bound ephrin-B2. (A-D) Swiss 3T3 fibroblasts in a starved, confluent monolayer were injected with 200 µg/ml pRK5-ephrin-B2 and 100 µg/ml biotin-dextran adjacent to cells co-injected with 100 µg/ml pCIneo-EphB4 and either 100 µg/ml pcDNA3-EGFP-APPPP-mito (A,B) or pcDNA-EGFP-FPPPP-mito (C,D) and left to express for 3.5 hours. Cells were then fixed and stained for biotin-dextran (blue) and p-Eph (red) to detect ephrin-B2- and EphB4-expressing cells, respectively; EGFP fluorescence (green) revealed expression of FPPPP-mito or APPPP-mito (A and C also show F-actin staining in green). The APPPP-mito-expressing cells showed internalisation of p-Eph (A,B). By contrast, p-Eph internalisation was inhibited in FPPPP-mito-expressing cells, with p-Eph staining instead remaining at sites of EphB4-ephrin-B2 contact (C,D). (E) Cells that expressed EphB4 and FPPPP/APPPP-mito, contacting ephrin-B2-expressing cells were scored for internalisation of p-Eph and the presence of large, invasive protrusions. FPPPP-mito expression significantly reduced the percentage of cells that internalised activated Eph receptors compared to APPPP-mito (P<0.05, n=5), with 193 (APPPP-mito) and 119 (FPPPP-mito) cells counted for each condition (five separate experiments). No difference was observed in the proportion of cells that displayed large, invasive protrusions. Bars, 10 µm.