Fig. 2.

PDCoV nsp15 impairs SEV-induced activation of NF-κB. (A–D) LLC-PK1 cells (A, C) or HEK-293T cells (B, D) grown in 24-well plates were co-transfected with NF-κB-Luc (A, B) or IRF3-Luc (C, D) and pRL-TK together with increasing quantities (0.1, 0.5, and 1.0 μg) of PDCoV nsp15 expression plasmid for 24 h, followed by infection with SEV or mock-infection for 12 h before luciferase reporter assays were performed. The averages of data from three independent experiments are shown. ***, p <  0.001. (E, F) LLC-PK1 cells (E) or HEK-293T cells (F) cultured in 60-mm dishes were mock-transfected or transfected with increasing amounts (3, 5, and 7 μg) of pCAGGS-HA-nsp15 for 24 h before infection or mock-infection with SEV for 8 h. Western blot analyses with antibodies against p-IRF3 (Ser386), IRF3, p-p65 (Ser536), p65, HA, or β-actin were performed on lysates from these cells. The relative levels of rate of p-p65 /p65 in comparison to the control group are shown as fold values below the images via Image J software analysis. (G, H) LLC-PK1 cells (G) or HEK-293T cells (H) cultured in 24-well plates were transfected with 1.0 μg of pCAGGS-HA-nsp15, followed by SEV infection for 6 h, and subjected to indirect immunofluorescence assays to detect endogenous p65 (green) and PDCoV nsp15 (red) with rabbit anti-p65, and mouse anti-HA antibodies, respectively. The cell nuclei (blue) were stained with DAPI. Fluorescent images were acquired with a confocal laser scanning microscope (Fluoviewver. 3.1; Olympus, Japan). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)