FIGURE 3.

LXXLL motif is not involved in the regulation of IRF3- and IRF7-mediated promoter activity. A, alignment of SAP domain of murine PIAS proteins. Conserved LXXLL motif and PIASy/mSAP mutation are boxed. B, PIASy^−/− MEF were transfected with WT PIASy or PIASy/mSAP. IFNβ was treated for indicated times, and mRNA for ISG15, IFI56, and IRF7 were quantified by qRT-PCR and by normalizing with hypoxanthine-guanine phosphoribosyltransferase mRNA. C, whole cell extracts in B were Western blotted (WB) with the indicated antibodies. D, 293T cells were transfected with ISRE reporter and increasing doses of WT PIASy or PIASy/mSAP. Cells were treated with IFNβ for 6 h, and luciferase activities were quantified by normalizing with Renilla luciferase activities. E, cells in B were infected with VSV for the indicated times, and IFNβ (left panel) and IFNα4 (right panel) mRNAs were quantified. F, 293T cells were transfected with VISA (left panel) or TRIF (right panel), IFNβ promoter reporter, and increasing amount of WT PIASy, PIASy/mSAP, or PIAS3. Luciferase activities were quantified as in D. G, cells in B were infected with VSV for the indicated times, and mRNAs were quantified as in B. H, 293T cells were transfected with VISA (left panel) or TRIF (right panel), ISRE reporter, and increasing doses of WT PIASy or PIAS/mSAP. Luciferase activities were quantified as in D.