SHIP-2 down-regulates Rac activation. Raw 264.7 cells were transiently transfected with either a nonspecific control siRNA or SHIP-2 siRNA. (A) 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 the indicated time. Protein-matched cell lysates were incubated with GST-PAK1-PBD-agarose beads for 1 hour at 4°C. Active Rac bound to the beads was eluted and loaded on 12% SDS-PAGE (top panel). Equal volume of lysis buffer only and whole cell lysate were loaded as negative and positive controls (neg and WCL). The membranes were reprobed with anti-GST antibody (middle panel). Rac band intensities were quantitated and are presented as fold increase over the resting control siRNA-transfected sample (bottom panel). The graph represents means ± SD. (B) Whole cell lysates from the resting samples were separated on 10% SDS-PAGE and transferred to nitrocellulose membrane, which was then probed with anti-SHIP-2 (top panel) or anti-SHIP-1 antibody (bottom panel).