Overexpression of SHIP1 blunts monokine-activated IFN-γ production in NK92 and primary human NK cells. (A-B) NK92 cells were retrovirally infected using the PINCO-GFP vector or PINCO vector encoding both SHIP1 and GFP cDNAs. (A) Infected cells were FACS sorted for GFP, and lysates were prepared. Protein lysates were next blotted with anti-SHIP1 and antiactin Abs. Lysates from the transfected Phoenix packaging cell line served as a positive control. (B) NK92 cells that were either infected with PINCO-GFP or with PINCO-SHIP1-GFP were each sorted for GFP⁺ cells and then incubated overnight with IL-12 (10 ng/mL) plus IL-18 (100 ng/mL), after which cells were assessed for IFN-γ protein production using intracellular flow cytometry. Results are representative of 2 experiments. (C-D) Enriched primary human NK cells were infected with PINCO-GFP or PINCO-SHIP1-GFP vectors. (C) Infected cells were FACS sorted for CD56 and GFP. (D) Sorted CD56⁺GFP⁺ NK cells were costimulated for 18 hours with IL-12 and IL-18, and supernatants were then harvested and quantified for IFN-γ production by ELISA. This experiment is representative of 7 performed with similar results. (E-F) Enriched primary CD56^bright NK cells were infected with PINCO-GFP or PINCO-SHIP1-GFP vectors. (E) Infected cells were FACS sorted for CD56^bright and GFP. (F) Sorted CD56^bright GFP⁺ NK cells were costimulated for 18 hours with IL-12 and IL-18, and supernatants were then harvested and quantified for IFN-γ production by ELISA. This experiment is representative of 3 performed with similar results. Error bars indicate ± SEM (see “Materials and Methods”).