IFN-γ production by FcR-stimulated NK cells in the presence of IL-12 is dependent upon activated Syk, PI-3K, and the ERK MAPK, but not p38, Jnk, or Akt. NK cells were pretreated with increasing concentrations of biochemical inhibitors of (A) Syk (piceatannol), (B) PI3-K (wortmannin), (C) ERK (U0126), or (D) Akt (Akt inhibitor; Calbiochem), and then plated in the immobilized IgG + IL-12 assay. Culture supernatants were harvested after 24 hours and analyzed for IFN-γ content by ELISA. Cells were counted via trypan blue exclusion following inhibitor pretreatment and following the IgG + IL-12 culture period to ensure equal viability of control-treated and inhibitor-treated NK cells. Pretreatment of NK cells with inhibitors of the p38 MAPK or Jnk MAPK had no effect on IFN-γ production in response to immobilized IgG and IL-12 (data not shown; similar results as with Akt inhibitor). (E) NK cells were pretreated with the indicated inhibitor and activated with various control stimuli (5 nM IL-2 or 50 ng/mL PMA plus 500 ng/mL ionomycin) for 10 minutes at 37°C. Cells were lysed, and levels of each phosphoprotein and total protein were measured by immmunoblot analysis. *P < .05 versus DMSO control for the IgG + IL-12 stimulation condition. Results represent the mean plus or minus SEM from n = 5 determinations.