SHIP-2 is recruited to the site of phagocytosis. Raw 264.7 cells 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 goat anti-SHIP-2 antibody followed by a Cy5-conjugated donkey anti-goat F(ab′)2 fragment (yellow). F-actin was stained with FITC-phalloidin (green), and nuclei were stained with Hoechst (blue). Coverslips were mounted on slides and read using Zeiss 510 confocal microscope with ×63 magnification. SHIP-2 has a diffused cytoplasmic pattern in resting cells (top panel). SHIP-2 localizes to the site of phagocytosis (middle panel), where it colocalized with F-actin. Cells stained with normal goat IgG followed by Cy5-conjugated donkey anti-goat F(ab′)2 fragment showed clean background (bottom panel).
