SH2 and PRD domains are both required for translocation of SHIP-2 to the site of phagocytosis. Raw 264.7 cells transfected with HA-tagged SHIP-2 constructs were grown on coverslips and either left untreated (resting) or incubated with IgG-coated SRBCs (red) for 5 minutes at 37°C. Cells were then fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100. After blocking, cells were stained with a mouse anti-HA antibody followed by an Alexa Fluor 594-conjugated goat anti-mouse F(ab′)2 fragment (yellow). Coverslips were mounted on slides and read using Zeiss 510 confocal microscope with ×63 magnification. (A) Empty vector-tranfected cells; (B) HA-SHIP-2 WT transfectants; (C) HA-SHIP-2 ΔSH2 transfectants; and (D) HA-SHIP-2 ΔPRD tranfectants.
