FIG 2.

NS6 inhibits activation of IRF3 and NF-κB. (A and B) HEK-293T cells were transfected with the indicated amounts of pCAGGS-HA-NS6 or empty vector, together with IRF3-Luc (A) or NF-κB-Luc (B) and pRL-TK plasmids, followed by stimulation with SeV, and they were analyzed as described in the legend to Fig. 1A. Anti-HA antibody was used to detect the expression of PDCoV NS6, and anti-β-actin antibody was used to detect β-actin protein by Western blotting (WB), which served as a protein loading control. (C and D) HEK-293T cells were transfected with pCAGGS-HA-NS6 or empty vector. After 24 h, the cells were infected with SeV or left untreated for 8 h. Cell lysates were collected for Western blot analysis with primary antibodies against phosphorylated IRF3 (p-IRF3 Ser386) and total IRF3 (C) or phosphorylated p65 (p-p65 Ser536) and total p65 (D), HA, and β-actin. (E and F) HEK-293T cells were transfected with pCAGGS-HA-NS6 or empty vector, followed by mock infection or SeV infection for 8 h as described for panels C and D. The cells were then fixed and subjected to an immunofluorescence assay with rabbit anti-IRF3 and anti-p65 and mouse anti-HA antibodies as primary antibodies, followed by staining with secondary antibodies Alexa Fluor 488-conjugated donkey anti-rabbit IgG (green) or Alexa Fluor 594-conjugated donkey anti-mouse IgG (red). DAPI staining (blue) indicates the locations of the cell nuclei. Fluorescent images were acquired with a confocal laser scanning microscope (FluoView ver. 3.1; Olympus, Japan). *, P < 0.05; **, P < 0.01; ***, P < 0.001.