Figure 10

Figure 10

The two basic residues are essential for inducible nuclear import of IRF3/5/7 by transfection of poly(I:C) (A) and TBK1 (B). (A) Mutation of the two basic residues impaired the inducible nuclear import of IRF3/5/7 by poly(I:C) transfection. HEK293T cells seeded overnight on microscope slide cover-glasses in six-well plates were transiently cotransfected as in Figure 4 for 24h, with the indicated IRF plasmids (2μg each) and poly(I:C) (100ng/ml). (B) Mutation of the two basic residues impaired the nuclear import of IRF3/5/7 by overexpression of TBK1. HEK293T cells seeded overnight on microscope slide cover-glasses were transiently transfected as in Figure 4, with the indicated IRF plasmids and TBK1 (2μg each) for different time points (24, 30, 48h), followed by a time-course confocal microscopy examination. Left panels showed the representative images at 30h post transfection. Right panels: The ratio of the nuclear-translocated cells to total fluorescent cells was statistically quantitated by cell counting of the whole visual field under confocal microscopy. (C) Immunofluorescence microscopy observation of endogenous IRF3 and IRF7 proteins in cells with or without stimulation by IFN stimuli. CO cells seeded overnight on microscope slide cover-glasses were transiently transfected with TBK1 (2 μg) or poly(I:C) (100ng/ml or infected wit SVCV or GCRV(1×10³ TCID₅₀/ml each), or mock treatment as control. 24h later, the cells were fixed, incubated overnight with polyclonal antibodies of fish IRF3 and IRF7, stained with fluorescent secondary antibody (Alexa Fluor Plus 555 TRITC). Red signal indicated endogenous IRF3 and IRF7 proteins. An enlarged view of the highlighted area in the last column display box. (D) Pull-down analysis of the binding affinity of Endogenous IRF3 and IRF7 proteins to fish promoter DNA. CO cells seeded overnight in 10cm² dishes were transfected with TBK1 or empty vector (5μg each) for 30h. The biotin-labeled DrIFNφ1 (-586 to +38) or DrIFNφ3 (-1447 to -910) promoter DNA (30 ng each) was incubated with the transfected cell lysates overnight at 4°C, followed by western blotting to detect IRF3 and IRF7 proteins by fish antibodies specific to IRF3 and IRF7. Note: the low binding intensities in empty vector-transfected CO cells might be due to the low level of constitutive expressed IRF3 and IRF7, which finally resulted in unsaturated protein binding to DNA in the pulldown assays.