Figure 4.

The Ndc80 complex interacts with Bod1 in mitotic HeLa cell extracts and in vitro. (a) Graphic representation of the full-length Ndc80 complex. Domains of interest, including the microtubule and centromere binding regions of the complex, are labelled. (b) Co-precipitation of Ndc80 complex components Ndc80, Nuf2 and Spc24 from mitotic HeLa cell extracts with purified Bod1-GST. (c) Representation of the recombinant Ndc80 Bonsai complex (as described in [38]). Ndc80–Spc25 and GST–Nuf2–Spc24 are expressed as fusion proteins. Both fusion constructs are co-expressed in E. coli from a dual-expression vector. (d) Of note, 150 pmol recombinant Ndc80 Bonsai, consisting of dimers of one Ndc80–Spc25 fusion protein with one GST–Nuf2–Spc24 fusion protein, was immobilized on Sepharose beads and incubated with 1 nmol Bod1-MBP or MBP. Binding was allowed for 1 h. Proteins were resolved by SDS-PAGE and immunoblotted using simultaneous detection of the MBP (red) epitope tag on Bod1 and the GST (green) epitope tag on Nuf2–Spc24. (e) Amount of bound protein in (d) was quantified relative to the input. Two asterisks indicate level of significance (p < 0.01) in unpaired Mann–Whitney rank sum test. n = 9 separate experiments. Error bars represent standard error.