Mapping of the binding regions between NFBD1 and PLK1.

<p>(A) A schematic drawing of the structure of NFBD1 is shown. Numbers indicate the amino acid positions relative to the first Met (+1). FHA, forkhead-associated; PST, proline/serine/threonine-rich; BRCT, BRCA1 carboxyl terminus. (B) <i>In </i><i>vitro</i> binding assay. Whole cell lysates prepared from COS7 cells transfected with FLAG-Plk1 expression plasmid were incubated with ³⁵S-labeled FHA, PST, or the BRCT domain of NFBD1. Bound materials were recovered by immunoprecipitation with an anti-PLK1 antibody and analyzed by SDS-PAGE followed by autoradiography (left panel). The right panel shows 1/10 loading. (C) The structure of wild-type Plk1 and various COOH-terminal deletion mutants of PLK1. KD, kinase domain; polo, polo box domain. (D) GST pull-down assay. ³⁵S-labeled wild-type PLK1 or the indicated deletion mutants of PLK1 were incubated with GST-BRCT fusion protein. After the incubation, bound proteins were recovered on glutathione Sepharose beads and separated by SDS-PAGE followed by autoradiography (left panel). The right panel shows 1/10 loading.