In vitro Mps1 Kinase Assay

For in vitro Mps1 kinase assay, immunoprecipitated endogenous Mps1 protein from C4–2 Neo, C4–2 D2 mitotic shake-off cells (cells were treated with 50 ng/ml nocodazole for 16 h) and asynchronous cells were incubated in Mps1 kinase reaction buffer (20 mM HEPES at pH 7.4, 100 mM KCl, 10 mM MgCl2, 1 mM EDTA, 0.5 mM DTT, 5% glycerol, 10 μM ATP and 0.17 μM γ−³²P ATP) with myelin basic protein (MBP) as substrates. The reaction took place in an incubator for 30 minutes at 30 °C and was stopped by adding sodium dodecyl sulfate (SDS) sample buffer. After the kinase reaction, samples were subject to SDS-polyacrylamide gel electrophoresis analysis. Incorporation of ³²P was determined by a Typhoon 9410 Imager (GE Healthcare Life Sciences) [32 (link)].