SHIP-2 down-regulates phagocytosis by suppressing upstream Rac activation. (A) Raw 264.7 cells were transiently transfected with constitutively active Rac (CA-Rac). Eight hours after transfection, cells were starved in incomplete RPMI media (ie, with no FBS) for 16 hours. After starvation, cells were stimulated with 2.4G2 followed by MAR (mouse anti-rat IgG) for 5 minutes to cluster FcγR. Protein-matched cell lysates were incubated with GST-PAK1-PBD-agarose beads for 1 hour at 4°C. Equal volume of lysis buffer was incubated with beads as a negative control (neg). Active Rac bound to the beads was eluted and loaded on 12% SDS-PAGE (top panel) and analyzed by Western blotting with anti-Rac antibody. The membrane was washed and reprobed with an anti-GST antibody (bottom panel). (B) Raw 264.7 cells were cotransfected with either a nonspecific control siRNA or SHIP-2 siRNA with CA-Rac. Twenty-four hours after transfection, whole cell lysates were made and separated on 12% SDS-PAGE and probed with an anti-Rac antibody. (C) Parallel samples were separated by 10% SDS-PAGE and probed with anti-SHIP-2 (top panel) or anti-SHIP-1 (bottom panel) antibody. (D) IgG-coated SRBCs were incubated with the transfectants for 1 hour at 37°C to assess phagocytic efficiency. Phagocytosis was measured by counting the total number of RBCs ingested by 100 phagocytosing cells. □ indicates control siRNA; ▪, SHIP-2 siRNA. The graph represents means ± SD.