Fig. 3. Effect of dominant-negative NIK mutants on Tax- and NIK-induced p100 processing. (A) 293 cells were transfected with Tax (1 µg) or HA-tagged NIK (0.8 µg) together with the indicated amounts of HA-tagged NIK(650–947) (NIK-C) or NIK(KK429–430AA) (NIK-KA). All the cells were also transfected with p100 (0.5 µg). The processing of p100 and expression of HA-tagged NIK proteins were analyzed by IB using anti-p100 (upper panel) and anti-HA (lower panel), respectively. Wild-type NIK and NIK-KA comigrate in the gel. (B) 293 cells were transfected as in (A) except that p100 was replaced with κB–TATA–luciferase and a control Rennila luciferase reporter driven by the constitutive thymidine kinase promoter. Dual luciferase assays were performed as described in Materials and methods. The κB-specific luciferase activity is presented as fold induction relative to the basal level measured in cells transfected with the pcDNA empty vector. The values shown are representative of three independent experiments.