Fig. 5. Ena/VASP proteins localise to sites of Eph receptor activation. (A) Schematic diagram of the neighbouring cell injection assay (see Marston et al., 2003): EphB4-expressing cells (green) make p-Eph-positive protrusions (red) under ephrin-B2-expressing cells (blue), which subsequently collapse rearwards as membrane ruffles. This leads to the internalisation of p-Eph and cell-cell separation around 3 hours post-injection. (B,C) Non-injected, starved Swiss 3T3 cells were stained for VASP (B) and Mena (C) to demonstrate localisation in the absence of Eph receptor activation. (D-G) Swiss 3T3 fibroblasts were injected with pCIneo-EphB4 (B4) or pRK5-ephrin-B2 (^*) and left to express for 2-3.5 hours, then fixed and co-stained for activated Eph receptors using anti-phospho-tyrosine (p-Y) or anti-p-Eph antibodies (red in merged images) and EphB4 (D,E), VASP (F) or Mena (G; green in merged images). EphB4 and p-Eph staining overlap at sites of EphB4-ephrin-B2 contact, including in some internalised vesicles (D,E). VASP (F) and Mena (G) localise to the EphB4/ephrin-B2 interface at sites of Eph receptor activation and are often found at the margins of protrusions made by EphB4-expressing cells at these sites. Bars, 20 µm.