FIGURE 3.

Leu¹⁹⁴ is critical for TICAM-1-mediated IRF-3 activation. A, L194A mutant lost IFN-β promoter-activating ability. HEK293 cells were transfected with increasing amounts of wild-type (WT) TICAM-1 or L194A mutant expression vector (1, 10, and 100 ng) with an IFN-β reporter and phRL-TK. Luciferase activity was measured 24 h after transfection. Representative data from a minimum of three separate experiments are shown. B, L194A mutant activates NF-κB and AP-1. HEK293 cells were transfected with wild-type TICAM-1 or L194A mutant expression vector (100 ng) together with NF-κB (left panel) or AP-1 (right panel) reporters. C, L194A lost IRF-3 activating ability. HEK293 cells were transfected with empty vector, wild-type TICAM-1 vector, or L194A vector (100 and 400 ng). After 24 h, lysates were prepared and subjected to native PAGE. Monomeric and dimeric forms of IRF-3 (arrowheads) were detected by Western blot. IB, immunoblot.