The S284 and T301 S/T-P motifs of Ana2 can be phosphorylated by recombinant Cdk1/Cyclin B kinase in vitro. (A) The sequence of Ana2 (aa278-306) highlighting the S/T-P motifs at S284 and T301. (B) The indicated biotinylated peptides were synthesised in vitro and incubated with ³²P-ATP in the presence of recombinant human Cdk1/Cyclin B, or buffer alone. The reaction mixtures were spotted onto nitrocellulose membranes and autoradiographs were obtained before the membranes were probed with anti-biotin antibodies to confirm the approximately equal loading of the peptides. The peptides including S284 and T301 were phosphorylated specifically in the presence of the kinase to approximately the same extent as the positive control peptide, and this phosphorylation was essentially abolished if S284 or T301 was mutated to Alanine. We conclude that both sites are strongly and specifically phosphorylated by Cdk1/Cyclin B in vitro. A representative blot is shown from three technical repeats.