NS4B decreases TRIF protein level.

<p>(<b>A-B</b>) HEK293T cells were co-transfected with the IFN-β-luciferease reporter plasmid, Flag-NS4B or NS3/4A expressing plasmid, together with TRIF or MAVS expressing plasmid for 24 h. The cells were harvested for luciferase assay <b>(A)</b> or for analysis by immunoblotting with anti-Flag, anti-NS3 and anti-Actin antibodies <b>(B)</b>. The luciferease activities were expressed as values relative to mock control. The error bars represent standard deviations from three independent experiments. One-way ANOVA was used for statistical analysis. ns, P>0.05; *P<0.05. (<b>C</b>) HEK293T cells transfected with plasmid expressing TRIF or MAVS and increasing dose of plasmid expressing Flag-NS4B (0, 100 and 200 ng) for 48 h were analyzed by Western blot using anti-Flag, anti-Actin antibodies. (<b>D-E</b>) HEK293T cells <b>(D)</b> or PH5CH8 cells <b>(E)</b> transfected with Flag-NS4B or NS3/4A expressing plasmid for 48 h were analyzed by Western blot using anti-TRIF, anti-MAVS, anti-Flag, anti-NS3 and anti-Actin antibodies. (<b>F</b>) Huh7 cells infected with HCVcc (MOI = 5) for the indicated time points were analyzed by Western blot using anti-TRIF, anti-Core and anti-Actin antibodies. The protein levels were quantified by Image J from three independent experiments, normalized against internal Actin control and expressed as values relative to the mock infection controls.