In vitro phosphorylation assay

In vitro phosphorylation assays were performed as previously described ^{18, 20}. In brief, bacterially expressed Cin8 (590)-TEV-EGFP-6His variants were purified using standard nickel affinity chromatography, eluted with 300mM imidazole that was subsequently removed using Zeba Spin Desalting Columns 40K (Thermo Scientific). For a phosphorylation assay, equal concentrations of Cin8 variants were mixed with TAP-purified Clb2-Cdk1- Cks1 complex in kinase assay mixture [50mM HEPES, pH 7.4, 150mM NaCl, 5mM MgCl₂, 8% glycerol, 0.2 mg/ml BSA, 500nM Cks1, and 500μM ATP (with added γ-³²P-ATP (PerkinElmer)]. Reactions were stopped after 10 and 20 min with SDS-PAGE sample buffer and proteins were separated by SDS-PAGE. Gels were stained with Coomassie Brilliant Blue (CBB) R-250 (Sigma) and incorporation of ³²P into the proteins was visualized by autoradiography.