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. 2000 Apr 11;97(9):4615–4620. doi: 10.1073/pnas.080583397

Figure 2.

Figure 2

Raf stimulates IKK-2 kinase activity. (A) IKK-2-induced NF-κB activity is enhanced by PMA and Raf. (A) Cotransfection of Jurkat T cells with 3xκB.luc and active Raf (Raf-BXB-CX) and/or wild-type IKK-2. Cells were stimulated with PMA as indicated. (B) IKK-2 overexpression results in constitutive phosphorylation of IκBα. (Upper) The autoradiograph of an immune-complex kinase assay using GST-IκBα as a substrate. Cells were transfected as above. VSV-IKK-2 was immunoprecipitated and incubated with either wild type (wt) or mutant IκBα (AA). Numbers indicate relative IκBα-specific kinase activities. Background activity in pcDNA3-transfected cell extracts was set to 1. (Lower) Control immunoblot analysis of immunoprecipitated IKK-2. (C) IKK-2 activity is increased by cotransfection of active Raf. Jurkat T cells were transfected with the indicated expression vectors. (Upper) An immune-complex kinase assay of immunoprecipitated VSV-IKK-2 with GST-IκBα as substrate. The asterisk marks the autophosphorylated IKK-2 protein. Numbers underneath the panel indicate the relative kinase activities. (Lower) Control immunoblot of precipitated IKK-2.