Biologic response of Stat1, Stat1K703R, and Stat1E705A in MEFs and macrophages. (A) IFN-γ–dependent antiviral response of Stat1, Stat1^K703R, and Stat1^E705A MEFs. Top panel: Stat1^–/– MEFs ectopically expressing empty vector (pMIG), Stat1 (St1), Stat1^K703R (St1^K703R), or Stat1^E705A (St1^E705A), as in Figure 4, were infected with VSV (MOI = 0.5) after IFN-γ (0, 0.5, 5, and 50 U/mL for 16 hours) pretreatment. Viral yield in supernatants of infected cells was determined, in triplicate, by plaque assay on Vero cells. Data are presented as total recovered PFUs (plaque-forming units). Bottom panel: viral yield from pMIG, St1, St1^K703R, and St1^E705A MEFs infected in triplicate with VSV at varying MOIs (0.001, 0.01, 0.1, and 1) after IFN-γ (5 U/mL for 16 hours) pretreatment. Viral titer was determined as for the top panel and is representative of 3 independent studies. (B) IFN-γ–dependent Stat1, Stat1^K703R, and Stat1^E705A activity in macrophages. Top panel: Stat1^–/– macrophages, infected with empty vector (pMIG), Stat1 (St1), Stat1^K703R (St1^K703R), or Stat1^E705A (St1^E705A), were evaluated for their capacity to produce NO 72 hours after stimulation with IFN-γ (50 U/mL) and/or LPS (2 μg/mL). Bottom left panel: surface MHC-II expression in GFP⁺ BMMs was determined by FACS 48 hours after stimulation with IFN-γ (50 U/mL). Bottom right panel: immunoblot demonstrates that Stat1 expression was similar in each set of transfectants. Data are representative of 3 independent studies. Error bars indicate standard deviation.