Caspase8 is required for the reduction of TRIF protein induced by NS4B.

<p>(<b>A</b>) HEK293T cells transfected with the plasmid expressing Flag-NS4B for 6 h were treated with DMSO control, chloroquine (20 μM) or Z-VAD-FMK (20 μM) for another 42 h, MG132 (5 μM) for 6 h. The cells were analyzed by immunoblotting with anti-TRIF, anti-Flag and anti-Actin antibodies. (<b>B-C</b>) HEK293T cells transduced with sgRNAs targeting caspase9, caspase8 or control (EGFP) were transfected with plasmid expressing Flag-NS4B. The cells were analyzed by immunoblotting with anti-TRIF, anti-Flag and anti-Actin antibodies. (<b>D</b>) HEK293T cells transfected with plasmids expressing Flag-NS4B or NS3/4A together with plasmids expressing TRIF-wt or TRIF-281E289E for 48 h were analyzed by immunoblotting with anti-TRIF, anti-Flag, anti-NS3 and anti-Actin antibodies. The TRIF protein levels were quantified by Image J from two independent experiments, normalized against internal Actin control and expressed as values relative to the mock infection controls.