Fig. 2.

Aurora-B phosphorylates Ser894 in the TSS motif of INCENP in vitro. (A) In vitro kinase assays were performed with either active (WT) or kinase-dead (KD) alleles Aurora-B proteins (kinases) and GST-fused INCENP-C-WT or -S894A protein (substrates) in the presence of [γ-³²P] ATP. Phosphorylated proteins were detected by autoradiography (³²P). Simply Blue staining was used as a loading control. (B) Aurora-B directly phosphorylates S894 of INCENP in vitro. In vitro kinase assays were performed as in A, except for the presence of [γ-³²P] ATP. Phosphorylation of INCENP was assessed by western blotting with the indicated antibodies. See Fig. S1 for uncropped gel images of SDS-PAGE (autoradiography and Simply Blue staining) in Fig. 2A. See Fig. S2 for uncropped western blot images in Fig. 2B. Black boxes indicate the cropped regions. Molecular sizes (kDa) are based on prestained protein markers.