FIG. 6.

Effects of dominant negative mutants of TRAF2 and TRAF6 on Tax induction of κB-luciferase activity. (A) 293 cells were transfected with 200 ng of κB-TATA-luciferase, 20 ng of 6RZ, and 0.1, 0.3, or 1 μg of a dominant negative FLAG-TRAF2(87-501) mutant. Approximately 24 h after transfection, the cells were stimulated with TNF-α (10 ng/ml) for 6 h. (B) 293 cells were transfected with 1 μg of the Tax expression vector, 200 ng of κB-TATA-luciferase, 20 ng of 6RZ, and 0.1, 0.3, or 1 μg of FLAG-TRAF2(87-501). Luciferase activity was determined approximately 30 h later and normalized to β-galactosidase activity. (C) HeLa cells were transfected with 200 ng of κB-TATA-luciferase, 20 ng of 6RZ, and 0.1, 0.3, or 1 μg of a dominant negative Myc-TRAF6(289-522) mutant. At approximately 24 h posttransfection, the cells were stimulated with IL-1β (10 ng/ml) for 6 h. (D) HeLa cells were transfected with 1 μg of Tax expression vector, 200 ng of κB-TATA-luciferase, 20 ng of 6RZ, and 0.1, 0.3, or 1 μg of Myc-TRAF6(289-522). Luciferase activity was determined at approximately 30 h posttransfection. The total amount of DNA in each transfection was kept constant at 4 μg by supplementation with pCMV4. Luciferase activity was normalized to β-galactosidase activity, and the values represent the means (± standard deviations) of three independent transfections. The values were first expressed as fold induction relative to cells transfected with reporters only. Final values were expressed as percentages of the TNF-α, IL-1, or Tax response.