Figure 2.

Activation of IKK-α and IKK-β by MEKK1 and NIK. HeLa cells were transfected with 1 μg of pRK-FlagIKK-α or 1 μg of pRK-FlagIKK-β and 6 μg of pCMV5-MEKK1, 6 μg of pcDNA3-NIK, or 6 μg of pcDNA3. Cells were harvested 42 hr after transfection, and IKK-α or IKK-β was immunoprecipitated with anti-Flag M2-agarose. (A) IKK immunocomplexes were assayed for kinase activity by incubation with 0.5 μg of GST-IκBα-(5–55) in the presence of [γ-³²P]ATP. Reaction products were subjected to SDS/PAGE in 10% gels and analyzed by autoradiography. The relative level of ³²P incorporation into the GST-IκBα-(5–55) substrate is indicated below the gel. (B) IKK immunocomplexes were subjected to SDS/PAGE in 8% gels, transferred to nitrocellulose membrane, and probed with anti-Flag polyclonal antibodies. (A and B) The positions of IKK-α, IKK-β, and GST-IκBα-(5–55) are indicated to the left, and molecular mass markers (in kDa) are indicated to the right.