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. 2018 Nov 22;37(24):e98658. doi: 10.15252/embj.201798658

Figure 2. IKK phosphorylates EDC4.

Figure 2

  1. In vitro kinase assay (KA) using endogenous IKKβ from cells, unstimulated or stimulated with IR (20 Gy, 45 min; top panel) or IL‐1β (10 ng/ml, 10 min; bottom panel) with purified GST‐EDC4 domains as indicated. Lower panel: cold KA as above, Coomassie blue staining. Asterisks denote specific bands.
  2. IKK phosphosite identification in EDC4 by mass spectrometry (see Table EV2 for MS data). Endogenous IKK purified from unstimulated or TNFα‐treated (10 ng/ml, 15 min) U2‐OS cells by immunoprecipitation of IKKγ was used in a cold KA with recombinant EDC4 sub‐regions, followed by MS analysis. Top, MS spectrum for phospho‐serine 583. Bottom, MS spectrum for phospho‐serine 855.
  3. In vitro KA of IKKβ (as in A) from TNFα‐stimulated cells with purified recombinant Strep‐EDC4 WD40 domain (EDC4 1–538) and point mutants for IKK phosphosites, S107A, S405A and S107/405A. Below: cold kinase assay.
  4. Diagram of EDC4 indicating IKKβ‐phosphorylated serines.

Source data are available online for this figure.