Phosphorylation of Dam1 by Ipl1–Sli15 prevents Ndc80 recruitment to microtubules. (A) The Ndc80 complex was tested for microtubule binding in the presence of phosphorylated and unphosphorylated Dam1 complex. (top) The phosphorylation experiments are outlined. The binding affinity curves plot averaged data from two independent experiments. Error bars represent SEM. (B) TIRF micrographs showing that the Dam1 complex phosphorylated by Ipl1–Sli15 fails to recruit the Ndc80 complex to dynamic microtubules (right). Bars, 2 µm. (C) The ability of the dam1 S4D (S20D, S257D, S265D, and S292) complex to recruit the Ndc80 complex to microtubules was tested in the presence of 100 mM NaCl. The dam1 S4D complex has a decreased ability to recruit Ndc80. The binding affinity curves plot averaged data from two independent experiments. SN, supernatant; P, pellet. Error bars represent SEM.