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. 2017 Jan 17;36(5):664–678. doi: 10.15252/embj.201694831

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© 2017 The Authors. Published under the terms of the CC BY NC ND 4.0 license

This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial‐NoDerivs 4.0 License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non‐commercial and no modifications or adaptations are made.

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Changes of the abundance of phosphorylated Mms4 peptides after Cdc5 inhibition (as in Fig 2C) (A, C) or in the absence of Cdc7 (B, D) in mitotically arrested cells.

A, B
Depicted are SILAC‐based intensity ratios of individual MS evidences for peptides of endogenously expressed Mms4. Evidences of non‐phosphorylated Mms4 peptides are shown in grey; evidences of phosphorylated peptides are shown in black, yellow, orange or blue. Blue colour indicates putative Cdc5 phosphorylation as defined by the (D/E/N)X(S/T) consensus (and additionally S268, which was also very strongly deregulated upon Cdc5 inhibition). Yellow or orange colours mark singly phosphorylated (S/T)(S/T) motifs, with orange marking p(S/T)(S/T) and yellow marking (S/T)p(S/T). Numbers indicate the phosphorylated residue in the depicted peptide. An asterisk marks peptide evidences that contained measured intensity values exclusively in the heavy or light sample. For doubly phosphorylated peptides, the two phospho‐sites are separated by a comma. For singly phosphorylated (S/T)(S/T) motifs, peptide ion fragmentation was in some cases unable to unambiguously identify the phosphorylated residue. In these cases, possible phosphorylation sites are indicated as “a/b”. Note that doubly phosphorylated (S/T)(S/T) sites were not reproducibly identified under conditions of endogenous Mus81‐Mms4 expression.

C, D
As in panels (A, B) but using Mus81‐Mms4 expressed from a high‐copy promoter. Depicted are SILAC‐based H/L ratios of individual MS evidences for phosphorylated peptides only. Peptides were sorted into categories according to their phosphorylation status: putative DDK target sites ((S/T)(S/T) motifs) were differentiated into the categories p(S/T)p(S/T) (red), p(S/T)(S/T) (orange) or (S/T)p(S/T) (yellow). Phosphorylated peptides matching the Cdc5 consensus site are coloured in blue. All other phosphorylated peptides are marked in grey. Bars depict the mean of the ratios of the respective category. Overall, Mms4 H/L ratio is shown on top.