Quantification of IFN-γ gene and protein expression in NK92 cells overexpressing SHIP1, or quantification of SHIP1 protein in NK92 cells, each following stimulation or costimulation by monokines. PINCO and PINCO-SHIP1–infected NK92 cells were stimulated for 18 hours with IL-12 (10 ng/mL), IL-18 (20 ng/mL), or IL-15 (100 ng/mL) and quantified for (A) induction of IFN-γ transcript by real-time RT-PCR or (B) secretion of IFN-γ protein by ELISA. Likewise, PINCO and PINCO-SHIP1–infected NK92 cells were stimulated for 18 hours with combinations of IL-12 and IL-15 or IL-12 and IL-18 and were quantified for (C) induction of IFN-γ transcript by real-time RT-PCR or (D) secretion of IFN-γ protein by ELISA. (E) Western blot analysis was performed on lysates of NK92 cells treated individually for 48 hours with IL-12, IL-15, or IL-12 and IL-18, or treated for 48 hours with IL-12 and IL-15 or IL-18, and then blotted with either anti-SHIP1 or antiactin Abs. This experiment is representative of at least 3 performed with similar results.