In vitro biochemical assays of the Sgt1–Hsp90 and Mis12 complex interactions. (A) HeLa cell extracts were subject to immunopurification (IP) after 48 h of treatment with control or Sgt1 siRNA and arrested for 16 h in nocodazole, using antibodies to Dsn1. The starting extract (L), the IP, and the post-IP supernatant (FT) were analyzed by immunoblotting using the indicated antibodies (labeled on the right side of the gel). (B) HeLa cell extracts were analyzed as in A, except that either Ndc80^Hec1 or CENP-K was used for the immunoprecipitation. The immunoblot in the top panel was probed with antibodies against both Ndc80^Hec1 and Hsp90. (C) The indicated proteins or Mis12 complex (referred to as Mis12) were purified, analyzed on SDS-PAGE, and stained with Coomassie dye. The migration position and cleavage products provide a reference for the same material used in the binding assays. The arrow labeled GST indicates the migration of cleaved GST; the arrow labeled prot indicates the migration position of the protease used to cleave GST from the fusion. *, the migration position of cleaved GST-Sgt1; **, the migration position of Sgt1 degradation. The diagram provides a key to complexes illustrated in the binding assays that follow. (D) The indicated µM concentration of GST-Sgt1 or GST were bound to glutathione beads and then used in the binding assay by adding Mis12 complex to 0.5 µM; complexes were identified that were associated with glutathione beads bound to the fusions. The diagram below the gel summarizes the binding observed, and the grayed subunits exhibit lower or nonspecific binding. Analysis of unbound fractions is included in Fig. S1. (E) GST-Hsp90 was preincubated with the indicated nucleotide (see Materials and methods) and then bound to the Mis12 complex in the presence or absence of Sgt1. Diagrams below the gels summarize the binding results. Numbers next to the gel blots indicate molecular mass in kD.