MT binding of the SAH domain is negatively regulated by phosphorylation. (A) Conservation of each residue in the PRDs relative to the mean conservation of residues in INCENP (top). Conservation was determined using conSURF (Landau et al., 2005) with 25 vertebrate INCENP homologs (Fig. S4 B). Putative Cdk sites conserved in at least 22 species are indicated (red circles). Diagrams of 6Myc-tagged constructs used in B, C, and D (bottom). (B) Western blot of various Myc-tagged xSAH domain constructs expressed in Xenopus egg extracts. Samples were either taken from extracts (–) or diluted in buffer containing lambda phosphatase and incubated at 30°C for 40 min (+). (C) Immunofluorescence of the indicated Myc-tagged constructs on the spindle in M-phase Xenopus egg extract (bottom). Western blot shows equal protein expression (top). Bar, 5 µm. (D) MT pelleting assay with the indicated constructs after depletion of endogenous INCENP. M-phase egg extract was incubated with nocodazole (gray circle) or taxol (black circles) for 30 min, and MT pellets were analyzed by Western blot. (E) MT pelleting assay as in D in extract reconstituted with the indicated GFP-xINCENP construct, xAurora B, xDasra A, and xSurvivin. Quantitation of pelleted GFP-xINCENP constructs relative to pelleted tubulin was performed in ImageJ and is shown at the bottom. (F) Western blot visualizing the MT-dependent activation of Aurora B. M-phase extract was reconstituted with the indicated INCENP constructs and then incubated in the presence of the indicated drug for 40 min. Aurora B activation was assessed by the appearance of Aurora B autophosphorylation (Aurora Bph) and OP18 hyperphosphorylation (OP18ph).