SHIP-2 down-regulation of FcγR-mediated phagocytosis is dependent on an intact SH2 domain as well as the C-terminal proline-rich domain. Raw 264.7 cells were transiently transfected with empty vector alone, HA-SHIP-2 WT, HA-SHIP-2 ΔSH2, or HA-SHIP-2 ΔPRD. GFP was cotransfected as a marker for transfection. (A) IgG-coated SRBCs were added to the transfectants 24 hours after transfection. The samples were incubated for 1 hour at 37°C to allow phagocytosis to take place. Cells were then subjected to brief hypotonic lysis prior to fixation in paraformaldehyde to be viewed under a fluorescence microscope. Phagocytosis was measured by counting the total number of SRBCs ingested by 100 transfectants (GFP-positive) each time for a total of 3 readings per sample in each experiment. ^*P < .05 compared with cells transfected with empty vector alone. The graph represents the mean ± SD. (B) Whole cell lysates from the transfectants were incubated with anti-HA antibody and protein G-agarose beads overnight. The precipitated proteins were next separated on 10% SDS-PAGE and transferred to nitrocellulose membrane, which was Western blotted with anti-HA antibody. The same membrane was washed and reprobed with anti-SHIP-2 antibody. These results are representative of 3 independent experiments.