For the pulldowns in Figures 1D and and3A,3A, Ndc80cdwarf with a 6-His affinity tag on the N-terminus of Ndc80 was immobilized to saturation on Co-NTA agarose by incubation with gentle agitation at 4°C. Beads were pelleted by centrifugation, washed three times in 20 mM HEPES pH 7.5, 200 mM NaCl, 2 mM imidazole pH 7.2, 2 mM β-mercaptoethanol, and 0.1% TWEEN-20 (wash buffer), and incubated with the relevant Stu2 construct for 30 min. Beads were again pelleted, washed three times with wash buffer, pelleted a final time, and the wash buffer aspirated from the tube. To elute bound proteins, 50 µL of wash buffer supplemented with 500 mM imidazole pH 7.5 was added to ~25 µL of beads. After an additional spin, the eluted proteins were visualized by SDS-PAGE. The pulldown in Figure 1B was carried out identically, with the exceptions that it used Ni-NTA agarose beads and that the wash buffer contained 150 mM instead of 200 mM NaCl.

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