Fig. 2. Ena/VASP proteins and Fyb/SLAP accumulate at sites of phagocytosis in primary macrophages. (A,C) Western blot analysis of protein extracts of monocyte-derived macrophages using an anti-VASP monoclonal antibody (A) and anti-Fyb/SLAP (C) affinity-purified polyclonal antibody. (B,D) Analysis of VASP and Fyb/SLAP intracellular distribution in monocyte-derived macrophages during phagocytosis. Cells undergoing phagocytosis were incubated with IgG-opsonised sheep RBC (for VASP labelling) or beads (for Fyb/SLAP labelling) for approximately five minutes, fixed and stained with the antibodies described above. In macrophages engaged in phagocytosis, both VASP (B) and Fyb/SLAP (D) localise to forming phagosomes. Again, both proteins colocalise with actin at these sites (B,D). Arrows point to examples of phagocytic cups surrounding sheep RBC (B) or beads (D). Scale bar: 10 µm.