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. 2016 Oct 10;215(1):77–93. doi: 10.1083/jcb.201603019

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© 2016 Redli et al.

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Figure 5. — The Ska complex promotes the catalytic activity of Aurora B in vitro. (A) Time course kinase assay with Aurora B–His and MBP–INCENP^790–919–His preincubated with Ska complex or equimolar amounts of BSA, as control, before addition of histone H3 and γ-[³²P]ATP. (B) Quantification of histone H3 ³²P signals from A. Signals were normalized to H3 and Aurora B protein levels monitored by Ponceau S staining (Ponc. S) and Western blotting (WB), respectively. Signal intensities are expressed relative to the first time-point. Data represent mean ± SD (three experiments). (C) Aurora B–His was incubated with recombinant Ska complex before pull-down with beads coupled to anti-Ska1 antibody or control antibody (IgG). UB, unbound fraction; B, bound fraction. (D) Immunoprecipitates (IP) from mitotic HeLa S3 cell extracts, obtained using anti-Ska1 antibodies or control antibodies (IgG), analyzed by WB. (E) Aurora B–His was preincubated with Ska complex (comp.) or histone H3, as control, before incubation with γ-[³²P]ATP. Aurora B autophosphorylation is visualized by autoradiography (³²P) and Aurora B levels by Coomassie Brilliant Blue (CBB) staining (see Fig. S5 G for uncropped results). (F) Quantification of Aurora B autophosphorylation signals from E (one experiment). Signal intensities are expressed relative to the first concentration. (G) Time course kinase assay with recombinant Aurora B–His preincubated with Ska complex, equimolar amounts of MBP–INCENP^790–919–His or BSA, as control, before addition of histone H3 and γ-[³²P]ATP. (H) Quantification of Aurora B kinase activity as in B (one experiment).