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MALDI-MS based enzymatic assay (data shown in Fig. 1)
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Kinetics were measured similar as reported before17. Full length kinase and pre-phosphorylated full length kinase were passed through SEC column on the day of the experiment to avoid soluble aggregates. The reaction mixture contained 100 µM p63 PAD peptide, prephosphorylated at S582, 50 nM kinase, 10 mM ATP, 1× Protease inhibitor, 0.5 mM TCEP in NMR Buffer. Kinetics were measured at 298 K. Aliquots of these reactions were quenched by the addition of 0.1% TFA at certain time points.
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