Fig. 10. (A) Analysis by western blotting of protein extracts of monocyte-derived macrophages and Jurkat T-cells with an anti-WASP monoclonal antibody. (B) Localisation of WASP in Raw264.7 cells undergoing phagocytosis. In macrophages engulfing IgG-opsonised beads, WASP colocalises with actin at phagocytic cups. Arrowheads point to sites where opsonised beads induced the formation of phagocytic cups. Stars indicate opsonised beads that have bound to a cell but have not induced the accumulation of actin or WASP. Scale bar: 10 µm. (C) Fyb/SLAP, SLP-76, Nck, Ena/VASP proteins and WASP are located in a multi-protein complex in stimulated Raw264.7 cells. Cell lysates from control and stimulated Raw264.7 cells were incubated with the immobilised WASP monoclonal antibody 67B4. Immunoprecipitates were resolved by SDS-PAGE, blotted and probed with antibodies to Fyb/SLAP, SLP-76, Nck and Evl. The stimulation of Raw264.7 cells results in the association of Fyb/SLAP, SLP-76, Nck and Evl with WASP. E, full Raw264.7 cell lysate; WASP IP, WASP immunoprecipitate.
