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. 1997 Sep 2;94(18):9792–9796. doi: 10.1073/pnas.94.18.9792

Figure 5.

Figure 5

Role of NIK in TNF- and TRAF2-induced NF-κB and JNK activation. Inhibition of TNF- (A and B) and TRAF2-mediated (C and D) activation of NF-κB but not JNK by NIK(KK429–430AA). 293 cells were transiently cotransfected with an E-selectin–luciferase reporter gene plasmid (lanes 1–12), an expression plasmid for HA epitope-tagged JNK1 (lanes 1–12) and vector control (lanes 1, 2, and 7), TRAF2 expression plasmid (1 μg; lanes 8, 11, and 12), and 1 μg (lanes 3, 5, 9, and 11) or 3 μg (lanes 4, 6, 10, and 12) of expression vectors for NIK(KK429–430AA) (lanes 3, 4, 9, and 11) or MEKK1Δ(K432M) (lanes 5, 6, 10, and 12). After 24 h, cells were stimulated for 30 min (A) or 6 h (B) with TNF (100 ng/ml) (lanes 2–6) or left untreated (lanes 1 and 7–12) before harvest. (A and C) Activity of HA epitope-tagged JNK1 was determined by immune complex kinase assay with GST–cJun(1–79) as a substrate. The location of the GST–cJun(1–79) fusion protein is indicated. The position of molecular mass markers (in kilodaltons) is shown on the left. (B and D) Luciferase activities corresponding to the transfections in A and C were determined and normalized based on β-galactosidase expression. Similar results were obtained in at least two independent experiments.