In vitro kinase assay

Transfected cells were lysed, and BubR1 was immunoprecipitated from lysates. Pelleted beads were washed with the immunoprecipitation buffer and then three times with the kinase buffer [20 mM HEPES (pH 7.8), 15 mM KCl, 10 mM MgCl2, 1 mM EGTA, and 0.1 mg/ml BSA] and then reacted with 20 μg of histone H1 in presence of unlabelled ATP or 20 μCi [γ³²P]ATP at 30^°C for 30 min. The reaction was stopped by adding SDS sample buffer. Proteins were resolved by 8% SDS-PAGE, and the results were quantified by autoradiography. For the in vitro phosphorylation of GST-BubR1, 1 μg of GST-BubR1 was washed twice with kinase buffer [20 mM HEPES (pH 7.8), 15 mM KCl, 10 mM MgCl2, 1 mM EGTA, and 0.1 mg/ml BSA] and reacted with 400 ng of recombinant Cdk1/Cyclin B1 or Plk1 (both from Invitrogen) in presence of unlabelled ATP (0.2 mM) at 30°C for 1 hr. Phosphorylated GST-BubR1 proteins were incubated with 5 μg His-Peli1 for 5 hr at 4^°C. Bead-bound protein complexes were washed twice with the immunoprecipitation buffer and analysed by immunoblotting.