FIGURE 6.

ALD-DNA induces dimerization/oligomerization of DAI and consequently activates DAI signaling pathways. A and B, dimer/oligomer formation of DAI by ALD-DNA stimulation. HA-tagged DAI (HA-DAI) and FLAG-tagged DAI (FLAG-DAI) were transiently coexpressed in HEK293 cells. The cells were stimulated with ALD-DNA (4 μg/ml) for the indicated periods (A) or stimulated with increasing amounts of ALD-DNA for 2 h (B) and analyzed by immunoprecipitation (IP) with anti-HA antibody, followed by immunoblotting with anti-FLAG (upper) and anti-HA (lower) antibodies. C, HEK293 cells were infected with 0.1 μg of pNF-κB-Luc, plus increasing amounts of pDAI, then left stimulated with UnALD-DNA (1 μg/ml) or ALD-DNA (1 μg/ml). Luciferase activities were measured and normalized to Renilla luciferase activities. D, HEK293 cells were infected with 0.1 μg of pNF-κB-Luc, plus 0.1 μg of pDAI, then left stimulated with increasing amounts of UnALD-DNA or ALD-DNA. Luciferase activities were measured and normalized to Renilla luciferase activities. E, HEK293 cells were infected with 0.1 μg of pIRF3-Luc, plus increasing amounts of pDAI, then left stimulated with UnALD-DNA (1 μg/ml) or ALD-DNA (1 μg/ml). Luciferase activities were measured and normalized to Renilla luciferase activities. F, HEK293 cells were infected with 0.1 μg of pIRF3-Luc, plus 0.1 μg of pDAI, then left stimulated with increasing amounts of UnALD-DNA or ALD-DNA. Luciferase activities were measured and normalized to Renilla luciferase activities. G and H, macrophages treated with siDAI or siControl were infected with 0.1 μg of pNF-κB-Luc (G) or pIRF3-Luc (H), plus 0.1 μg of pDAI, then left stimulated with UnALD-DNA (1 μg/ml) or ALD-DNA (1 μg/ml). Luciferase activities were measured and normalized to Renilla luciferase activities. Data are representative of results obtained in three independent experiments.