FIG. 7.

N protein does not affect expression level of and activity of viral P protein to inhibit the IRF-3 pathway. (A) SYM-I cells were infected with the Ni, Ni-CE, or CE(NiN) strain at an MOI of 2. After 24 h, the cells were lysed and N, P, and tubulin were detected by Western blotting. (B) SYM-I cells in a 24-well plate were cotransfected with 1 μg of each plasmid driving the expression of the indicated viral protein or empty vector. At 48 h posttransfection, the cells were lysed, and N, P, and tubulin were detected by Western blotting. (C) SYM-I cells in a six-well plate were cotransfected with 4 μg of pCAGGS-CEP and 4 μg of pCAGGS-NiN or pCAGGS-CEN. Cell extracts were prepared at 48 h posttransfection and directly subjected to Western blotting with anti-N antibody, anti-P antibody, or anti-tubulin antibody (top). The same cell extracts were subjected to coimmunoprecipitation analysis with anti-N antibody (middle) or normal mouse IgG (bottom). The immunoprecipitated samples were examined by Western blotting. (D) SYM-I cells were cotransfected with pRL-TK, 4×IRF-3-Luc, and 1 μg of each plasmid driving the expression of the indicated viral protein or empty vector. At 24 h posttransfection, the cells were infected with NDV at an MOI of 1 and incubated for 12 h. Then the cells were lysed and luciferase activities were measured. The data represent firefly luciferase activity normalized to Renilla luciferase activity and are presented as means (± the SD) of three independent replicates.