In vitro kinase assay

Kinase dead PLK1-K82R, GST-Bora, and GST-Aurora-A were purified from bacteria as described (Macurek et al, 2008) and incubated with CycA2-CDK2 (100 ng/reaction; Biaffin GmbH) in a kinase buffer (25 mM MOPS pH 7.2, 12.5 mM glycerol 2-phosphate, 25 mM MgCl₂, 5 mM EGTA, 2 mM EDTA, and 0.25 mM DTT) supplemented with 100 μM ATP and 5 μCi 32P-γ-ATP at 30°C for 30 min. After separation of proteins by SDS–PAGE, phosphorylation was detected by autoradiography or by pT210-PLK1 antibody.