Figure 3

Effect of the 3CD protein on the nuclear translocation of the IRF7 protein.A Three experimental groups were set up, both groups I and II were co-transfected with 0.3 µg pCAGGS-IRF7-Flag and 0.5 µg pCAGGS; the third group was transfected with 0.3 µg pCAGGS-IRF7-Flag and 0.5 µg pCAGGS-3CD-HA, and transfect the plasmids into cells in a 24-well plate. 24 h after transfection groups II and III were stimulated with 5 µg/µL Poly (I:C) for 12 h, and cell samples were collected for indirect immunofluorescence experiments. B Images such as those in panel A were subjected to image analysis (see Materials and methods), where > 30 cells for each sample were analyzed to determine Fn/c, the fluorescence in the nucleus relative to that in the cytoplasm. Results are means ± standard errors of the mean (n ≥ 30). C DEFs were cultured in 6-well plates, grouped and transfected as described above for group A (transfection ratio of 3:2), 24 h after transfection groups 2 and 3 were stimulated with 20 µg/µL Poly(I:C) for 12 h, and cell samples were collected for Western blot analysis. D Gray value analysis was performed on the bands in C with ImageJ software to determine the ratio of Pn/c: Pn/c = Pn/Pc, Pn (relative protein level of IRF7 in the nucleus) = gray value of nuclear IRF7 protein/ Histone H3.1, Pc (relative protein level of IRF7 in the cytoplasm) = gray value of cytoplasmic IRF7 protein/β-tubulin. Where the data in each group were normalized relative to the control group. Values represent the mean (± SD) from three independent experiments. NS: not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, compared with the control group.