SHIP-2 knockdown by siRNA results in enhanced FcγR-function. (A) Raw 264.7 cells were transiently transfected with (1) control siRNA, (2) SHIP-2 siRNA1, and (3) SHIP-2 siRNA2. Cells were harvested 24 hours after transfection, and protein-matched lysates were analyzed by Western blotting with anti-SHIP-2 antibody (upper panel) and anti-SHIP-1 antibody (lower panel). The middle panel is a quantitative measurement of SHIP-2 band intensities. (B) Raw 264.7 cells were transiently transfected with control siRNA or SHIP-2 siRNA1. Twenty-four hours after transfection, the IgG-coated SRBCs were added. The samples were incubated for 1 hour at 37°C to study phagocytosis. 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 RBCs ingested by 100 transfectants. ^*P < .05. (C) Raw 264.7 cells transfected with control siRNA or SHIP-2 siRNA were incubated with IgG-coated SRBCs for 1 hour at 4°C to study binding. Cells were then subjected to brief washing prior to fixation in paraformaldehyde to be viewed under a fluorescence microscope. The binding activity was expressed as the total number of bound SRBCs on 100 rosetting Raw cells that each bound 3 or more SRBCs (binding index). Graphs represent means ± SD.