The effects of wild-type PIAS1 and PIAS1-SID on Stat1-mediated gene activation in response to IFN-γ treatment. (A) Schematic representation of PIAS1 and PIAS1-SID constructs. (SID) Stat1-interaction domain (amino acids 392–541). (B) Luciferase assays. 293T cells were transiently transfected with [(3x)Ly6] luciferase reporter plasmid together with empty expression vector, Flag-Stat1 (1.0 μg) or various amounts of Flag-PIAS1 (0.1, 0.3, or 0.6 μg) or Flag-PIAS1-SID (0.5, 1.0, or 2.0 μg), alone or in combination as indicated. Cells were either untreated or treated with IFN-γ for 6 h followed by luciferase measurement. Shown is the -fold increase after IFN-γ treatment. (C) Western blot analysis. Protein extracts from equal numbers of transfected cells (corrected by relative expression of β-galactosidase) in B were analyzed by immunoblot with anti-Flag (Sigma). IFN-γ-treated samples in exactly the same order as in B were shown.