Effect of catalytic mutant SHIP1 (D675A) overexpression on IFN-γ production induced by monokine costimulation of NK cells. (A) Enriched primary NK cells were retrovirally infected using insertless PINCO, the PINCO vector encoding wild-type SHIP1, or the PINCO vector encoding the catalytic mutant D675A SHIP1. Infected cells were FACS sorted for CD56 and GFP, plated in medium, and costimulated with IL-12 plus IL-18 for 18 hours. (B) Supernatants were then collected and assayed for IFN-γ by ELISA. An immunoblot shows levels of SHIP1 and GRB2 in cell lysates from NK92 cells infected with insertless PINCO (lane 1), the PINCO vector encoding wild-type SHIP1 (lane 2), or the PINCO vector encoding the catalytic mutant D675A SHIP1 (lane 3). This experiment showed no significant decrease in IFN-γ production between primary NK cells infected with insertless PINCO and NK cells infected with the PINCO D675A SHIP1 vector and is representative of 3 experiments performed with similar results. Error bars indicate ± SEM.