Influence of Thr892 and Ser893 phosphorylation by Aurora-B on phosphorylation of INCENP-Ser894. (A) In vitro kinase assays were performed using recombinant LATS2 and/or Aurora-B and purified GST-fused INCENP-C-WT, -T892A, -S893A, -T892A/S893A (AAS), or -T892A/S893A/S894A (AAA) protein as a substrate, followed by western blotting with the indicated antibodies. Bar graphs show the relative levels of pS894 normalized against the corresponding band intensity of GST-INCENP. (B) In vitro kinase assays were performed using recombinant Aurora-B-WT (wild-type) or KD (kinase dead) as the enzyme and purified GST-fused INCENP-C-WT, -T892A, -S893A, -T892A/S893A (AAS), or -T892A/S893A/S894A (AAA) protein as the substrate, followed by western blotting with the indicated antibodies. (C) Proposed model for the influence of phosphorylation of neighboring sites (T892 and S893) on S894 phosphorylation in the presence of Aurora-B alone (i), LATS alone (ii), LATS alone + INCENP with T892A/S893A mutations (iii), and both Aurora-B and LATS (iv). See Fig. S2 for uncropped western blot images in Fig. 3A, B. Black boxes indicate the cropped regions. Molecular sizes (kDa) are based on prestained protein markers.