FIGURE 3:

CK2 directly phosphorylates Mif2 and Ndc10 in vitro. Mif2-Myc and Ndc10-Myc were overexpressed and affinity-purified as described previously. A stringent wash buffer containing 1 M KCl was used to remove Mif2-associated proteins prior to the kinase assay. The Dam1 complex purified from E. coli was used as a control. CK2 and GST-Ipl1 were purified from yeast and E. coli, respectively. Substrates (∼100 ng) were incubated with [γ³²P]ATP in the presence of kinases (∼2 ng). Top, autoradiography; bottom, Coommassie Brilliant Blue staining.