Fig. 2

CaIRF1 activates the IFNβ and ISRE promoters. A Western blot to determine CaIRF1 expression. p3 × Flag-CaIRF1 (0.5 μg) or empty plasmid (0.5 μg) was transfected into cells cultured on 24-well cell plates, and 24 h later, western blot was carried out to verify the expression of CaIRF1. GAPDH was used as the loading control. B IFA and GFP tag observation verified that CaIRF1 was expressed and localized mainly in the nucleus. Adjustments of individual colour channels are sometimes necessary on merged images. This part of the experiment was photographed under a confocal microscope (LEICA, TSC-SP8). Scale bar, 25 μm. C A dual-luciferase assay was performed to detect the activation of IFNβ and ISRE promoters induced by CaIRF1. The specified vector (0.5 μg) was cotransfected with the promoter plasmid (0.5 μg) and pRL-TK (0.025 μg). Twenty-four hours post-transfection, cells were stimulated with the poly(I:C) for 12 h or with no treatment and then assayed for IFNβ and ISRE promoter activity. D Results showing the effect of CaIRF1 regulation on the expression of ISG mRNAs (ISG15 and MxA). E MDCK cells were transfected with si-NC or si-CaIRF1 (40 pmol) for 36 h. Finally, the expression of endogenous CaIRF1 was measured by western blot. GAPDH was used as a loading control. F Knocking down CaIRF1 blocked the poly(I:C)-induced expression of ISGs. MDCK cells were transfected with si-NC or si-CaIRF1 (40 pmol) for 36 h. The cells were stimulated with the addition of poly(I:C) for 12 h, and then, the transcription of ISGs was detected by qPCR. The data shown in the figure represent the mean ± SD of three independent experiments, each experiment had three cell wells as technical replicates. ** P < 0.01, **** P < 0.0001 compared with the empty vector transfection group. The original images of the western blot and enlarged versions of the cell images were shown in the Supplementary file 1