FIG. 7.

Kinase-deficient IKKα blocks MEKK1- and Cot/Tpl-2-induced, but not PKCθ-induced, phosphorylation of IKKβ. HeLa cells and 293 cells were transfected with 1 μg of IKKβK44A-Flag expression plasmid and 1 μg of HA-tagged wild-type or kinase-deficient IKKα in the presence or absence of the Myc-Cot (A and B), HA-MEKK1 (C and D), and NIK and PKCθ(A/E) (E) expression vectors. After 24 h (293) and 48 h (HeLa), cells were harvested and lysates were immunoprecipitated with anti-Flag M2-agarose (A to D) or with IKKγ-specific antibodies (E). The immunoprecipitated complexes were subjected to an in vitro kinase assay and analyzed as for Fig. 4. The levels of phosphate incorporated into Flag-tagged, kinase-deficient IKKβ are shown in the upper panel, and the levels of Flag-tagged IKKβ are shown in the lower panels.