In vitro kinase assays

1.5 µg purified “kinase-dead” N-terminally 6His-tagged D274N Aurora A was incubated with 3 µg of N-terminally 6His-tagged WT Plk4 (1–285) lacking the polo box domains or a catalytically inactive D154A Plk4 (1–264) mutant (±10 µM centrinone) at 37°C in 50 mM Tris, pH 7.4, 10 mM MgCl₂, 1 mM DTT, and 1 mM ATP. Aliquots were removed at 0, 10, 30, 60, 120, and 240 min after reaction initiation and stopped by boiling in SDS sample buffer. To evaluate site-specific Aurora A phosphorylation, trypsin proteolysis and liquid chromatography–tandem mass spectrometry (LC-MS/MS) analysis was performed at the 120-min time point.