(A) The binding of phosphorylated CENP-T to MIS12CNano, a version of MIS12C designed for structural analysis (Petrovic et al., 2016), is abolished by the mutation of Tyr8, Phe12, and Phe13 to alanine in the MIS12 subunit. (B) Fluorescent polarization was used to determine the displacement of CENP-C1-21 from MIS12CNano upon the addition of either CENP-C1-71 or, at higher concentrations, CENP-T195-215. (C) Analytical size-exclusion chromatography (Superdex 200 5/150) shows that phosphorylated CENP-T is displaced from MIS12C when incubated with CENP-C, but not when incubated with a version of CENP-C that cannot bind to MIS12C. Fluorescently labeled CENP-T was monitored specifically during chromatography. Red asterisks indicate K10A and Y13A mutations in CENP-C. See Figure 7—figure supplement 2 for the binding of CENP-C and CENP-CK10A Y13A to MIS12C.