The two basic residues are essential for IRF1/2/11 to regulate IFN response. (A) Mutation of the two basic residues impaired the constitutively nuclear accumulation of IRF1/2/11. Left panels: HEK293T cells seeded overnight on microscope slide cover-glasses in six-well plates were transiently transfected as in Figure 4, with indicated IRF plasmids (2μg each) for 24h, followed by confocal microscopy examination. The last column showed magnification view of the area highlighted in the box. Right panels: The intensities of nucleus/cytoplasm GFP were quantitated using the ImageJ processing program and normalized to that of the empty construct pEGFP-N3, which was set to 1:1. (B) Pull-down analysis of the binding affinity of IRF1/2/11 wild types and IRF1/2/11 mutants to IFNφ1/IFNφ3 promoters. DNA pull-down assays were performed as in Figure 3B, by incubating biotin-labeled IFNφ1 or IFNφ3 promoter DNAs with appropriate amounts of HEK293T cell lysates, where cells were transfected for 30h with wild types or mutants of IRF1/2/11, respectively. (C, D) IRF1/11 mutants failed to stimulate the activation of crucian carp IFN promoter (C), and zebrafish IFNφ1/IFNφ3 promoters (D). EPC cells seeded in 48-well plates overnight were transfected with CaIFNpro-luc (C), IFNφ1pro-luc or IFNφ3pro-luc (D), together with the indicated IRF plasmids (100ng each) for 30 h, followed by luciferase assays. (E) IRF2 mutant failed to inhibit poly(I:C)-triggered activation of crucian carp IFN promoter by luciferase assays. EPC cells were transfected with the indicated plasmids (100ng each), together with poly(I:C) (1μg/ml) for 30h. (***P < 0.001, *P < 0.05).
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