Biochemical properties of Aust and Borr. (A and B) In vitro coprecipitation experiments. Extracts of mammalian cells expressing HA-Borr, HA-Aust, HA-DmINCENP, or FLAG–aurora B were incubated with either MBP, MBP-Borr, or MBP-Aust immobilized on amylose beads. Beads were washed and analyzed by Western blotting using anti-HA or anti-FLAG antibodies. (A) HA-Borr and HA-Aust coprecipitate with purified MBP-Borr and MBP-Aust but not with MBP alone. (B) FLAG-DmINCENP but not HA–aurora B coprecipitates with purified MBP-Borr and MBP-Aust. Input, mammalian cell extracts expressing HA- or FLAG-tagged constructs. +/− corresponds to the presence (+) or absence (−) of MBP fusion proteins. (C) In vitro DNA binding assay. Purified MBP fusion proteins were incubated with DNA–cellulose beads and assayed for binding by SDS-PAGE. MBP-Borr and MBP-Aust but not MBP alone bind to DNA–cellulose beads. Input, purified proteins before incubation; bound, proteins bound to beads after incubation and washing. Numbers indicate molecular mass in kD.