Supplementary Figure 5: Cyclin C-CDK19 and C-CDK3 kinases phosphorylate T2512, S2514 and S2517 of ICN1.

Cyclin C-CDK19 or cyclin C-CDK3 complexes purified from Sf9 cells were incubated with N-EHPFLTPSPESPDQWFPK-C peptide, corresponding to aminoacids 2507 to 2521 of Notch1, as described in Methods. Separation of these Notch1 peptides by liquid chromatography after in vitro kinase reactions demonstrated three distinct peaks, each representing one of the three in vivo identified Notch1 phosphorylation sites (T2512, S2514 and S2517). Each of these identified phosphorylation sites could be confirmed by MS/MS analysis and sites localized using an Ascore localization score. Representative MS/MS scans are shown for both cyclin C-CDK19 and cyclin C-CDK3 experiments.