Figure 6: Constitutive phosphorylation of S185/T205 perturbs MT binding of the Ska1–MTBD in vitro.

(a) Cartoon representation of Ska1–MTBD, where consensus Aurora B phosphorylation sites are shown in stick representation (red). The phosphorylation sites of Aurora B are located within two of the three main clusters responsible for the MT-binding activity of Ska1 (circled). (b) Representative SDS–PAGE of the MT cosedimentation assays where phosphomimic mutants S185D and S185/T205D are tested. (c,d) Left, schematic representation of both protocols used for the treatment of Ska or Ndc80 complex with Aurora B. Middle, representative SDS–PAGE of the cosedimentation assays, where (c) the Ska/Ndc80 complex was incubated first with Aurora B kinase and subsequently with MTs. (d) MT-bound Ska/Ndc80 complex was incubated with Aurora B. Right, quantification of the results obtained (mean±s.d., n3, *P0.05, ***P0.001; t-test). Uncropped scans of the gels are shown in Supplementary Fig. S8d.