Molecular mechanisms for Spi-B–mediated type I IFN promoter activation. (A) 293T cells were transiently transfected with an Ifna4 or Ifnb1 promoter-driven luciferase reporter plasmid alone or together with a combination of expression vectors for Spi-B (5.25, 10.5, 21, 42, or 84 ng/well) and IRF family members (8.4 ng/well). After 18 hours, cell lysates were prepared and subjected to the luciferase assay. (B) Interaction of Spi-B with IRF family members in 293T cells. IP indicates immunoprecipitation; IB, immunoblot; and WCL, whole cell lysate. (C) 293T cells were transiently transfected with an Ifna4 or Ifnb1 promoter-driven luciferase reporter plasmid alone or together with a combination of expression vectors for Spi-B mutants (21 or 84 ng/well) and IRF-7 (8.4 ng/well). The luciferase assay was carried out as indicated in panel A. Filled boxes denote HA tag. FL indicates full-length Spi-B; ΔTA, transactivation domain deletion mutant of Spi-B; ΔEts, Ets domain deletion mutant of Spi-B; and PEST, proline, glutamic acid, serine and threonine rich domain. (D) Interaction of Spi-B mutants with IRF-7 in 293T cells. (E) The following mutant promoters were used for the luciferase assay. Ifna4-am4Ets, the Ifna4 promoter carrying the mutation in all 4 Ets-binding sites; Ifna4-am4Ets9596, the Ifna4 promoter carrying the mutation in all 4 Ets-binding sites and an IRF-binding site; and Ifnb1-bm4Ets, the Ifnb1 promoter carrying the mutation in all 4 Ets-binding sites. Data are representative of 2 independent experiments (means ± SD in panels A, C, and E).