In vitro kinase assay and mass spectrometry

Kinase assays were carried out using 0.1 μg of purified NEK6 or NEK7 kinase (Millipore). Proteins were incubated with 5 μg of substrate and 1 μCi of [γ-32P]-ATP in 40 μl of kinase buffer [50 mM Hepes-KOH (pH 7.4), 5 mM MnCl₂, 5 mM β-glycerophosphate, 5 mM NaF, 4 μM ATP, and 1 mM dithiothreitol (DTT)] at 30°C for 30 min. Reactions were stopped with 50 μl of protein sample buffer and analyzed by SDS-PAGE and autoradiography. Phosphomapping was performed using an LTQ Orbitrap Velos ETD (Thermo Fisher Scientific) as previously described (35).