Fig. 5.

Recombinant LegK1 directly phosphorylates IκBα in vitro. (A) In vitro phosphorylation of IκBα by recombinant LegK1 and its truncation mutants. Purified GST-IκBα or myelin basic protein (MBP) was used as the substrate in the in vitro kinase assay by using indicated purified kinases. ³²P autoradiography (Upper) shows incorporation of phosphates into the substrate and immunoblotting using the pSer32-IκBα antibody (middle) reflects the site-specific phosphorylation. Lower shows the relative level of LegK1 added into each reaction. (B) NF-κB luciferase assays of LegK1 kinase activation loop mutant (SY/AA, LegK1 S252A/Y256A) (B) or truncation mutants of LegK1 (D). HEK 293T cells were transfected with LegK1 or its variants as indicated, and the relative NF-κB luciferase activity is shown. (C) Phosphorylation of IκBα induced by truncation mutants of LegK1 in HeLa S100 extracts.