IFN-γ–dependent expression of target genes in Stat1, Stat1K703R, and Stat1E705AMEFs. (A) Total RNA was prepared from Stat1^–/– MEFs ectopically expressing empty vector (pMIG), Stat1 (St1), Stat1^K703R (St1^K703R), or Stat1^E705A (St1^E705A), as in Figure 4, after stimulation with IFN-γ (50 U/mL for 0-12 hours), as indicated. The expression of target genes (IRF1, TAP1, GBP1, and Mig) was determined by quantitative PCR (Q-PCR) from cDNA templates. The relative expression of each gene was normalized to β-actin expression. (B) Stat1^–/– MEFs, ectopically expressing empty vector (pMIG), Stat1 (St1), Stat1^K703R (St1^K703R), or Stat1^E705A (St1^E705A), were transiently transfected with a GAS-driven luciferase reporter (B2SH-WT3-Luc) in triplicate and stimulated with IFN-γ (50 U/mL for 6 hours). Samples were harvested and evaluated for luciferase and renilla activity (in arbitrary light units). Data are representative of 2 independent experiments. Error bars indicate standard deviation. (C) Immunoblot demonstrates that Stat1 expression (wild-type and mutants) was similar in all 3 Stat1-expressing lines.