FIG. 8.

Specificity of NF-κB inhibition by Cot mutated at S400. (A) 293T cells were transfected with Flag-tagged IKKα and the indicated myc-tagged Cot or NIK constructs. Twenty hours after transfection cells were harvested, lysed, and subjected to immunoprecipitation with anti-Flag antibody M2. IKK kinase assays were conducted as described in Materials and Methods (upper gel). Aliquots of the lysates were assayed for expression of the myc-tagged proteins (lower gel). Equivalent levels of IKKα were confirmed by Western blotting (data not shown). (B and C) Dominant-negative activity of Cot SA(2). Jurkat T cells were transfected with an NF-κB-luciferase reporter and the indicated constructs. Cells were then stimulated for 6 h with 20 ng of PMA/ml (B), anti-CD3/CD28 (1 μg of anti-CD3/ml and 2 μg of anti-CD28/ml) (C), or 50 ng of TNF-α/ml (B and C), after which luciferase activity was determined. The results shown in panel C are shown as the percent maximal stimulation (versus vector control) and are the averages ± standard deviations of three experiments. WT, wild type; Autorad., autoradiography; WCL's, whole-cell lysates.