FIGURE 2.

TOPK phosphorylates IκBα at Ser-32. A, in vitro kinase assay was performed with GST-IκBα (0.5 μg) and each active TOPK (25, 50, and 100 ng). After 30 min of incubation at 30 °C, the reaction mixture was subjected to 10% SDS-PAGE and immunoblot (IB) using phospho-IκBα (Ser-32) antibody. B, TOPK kinase assay was done using active or inactive TOPK (each 100 ng). The phosphorylated IκBα was detected with phospho-specific antibody recognizing Ser-32 residue on IκBα. C, 293 cells were transfected with V5-TOPK or empty vector. At 24 h post-transfection, the cells were treated with doxorubicin (Dox, 1 μm) for 2 or 4 h, and cell lysate was immunoprecipitated (IP) with V5 antibody. The immunoprecipitation kinase assay was performed using GST-IκBα (0.5 μg).