Fig. 2.

DAK inhibits MDA5- but not RIG-I-mediated activation of ISRE and the IFN-β promoter. (A and B) DAK inhibits MDA5-mediated activation of ISRE (A) and the IFN-β promoter (B) in a dose-dependent manner. The 293 cells (1 × 10⁵) were transfected with ISRE (A) or the IFN-β promoter (B) luciferase reporter plasmid (0.05 μg), an expression plasmid for Flag-MDA5 (0.4 μg, gray bars) or an empty control plasmid (0.4 μg, white bars) and increased amounts of an expression plasmid for HA-DAK (0–0.8 μg as indicated). Luciferase assays were performed 16 h after transfection. (C–E) DAK has no significant effects on activation of ISRE and the IFN-β promoter mediated by RIG-I, TLR3, and VISA. The 293 cells (1 × 10⁵) were transfected with the indicated reporter plasmid (0.05 μg) and expression plasmids (0.4 μg of each). Reporter assays were performed 16 h after transfection. In E, transfected cells were treated with poly I:C (40 μg/ml) for 6 h before reporter assays were performed. (F) DAK inhibits MDA5-N- but not RIG-I-N-induced expression of IFN-β. The 293 cells (5 × 10⁵) were transfected with Flag-MDA5-N or Flag-RIG-I-N plasmid (1 μg) and HA-DAK or empty control plasmid (2 μg). RT-PCR was performed with human IFN-β or β-actin primers 18 h after transfection. (G) DAK does not inhibit MDA5-N and RIG-I-N-mediated activation of NF-κB. The 293 cells (1 × 10⁵) were transfected with NF-κB luciferase reporter (0.1 μg) and the indicated plasmids (0.4 μg of each). Reporter assays were performed 16 h after transfection. (H) DAK-N was sufficient to inhibit MDA5-mediated activation of ISRE and the IFN-β promoter. The experiments were performed as in C and D. Relat. Lucif. Act., relative luciferase activity; Vec, vector.