We re-introduced Flag-mTRADD into Tradd-/- MEFs, and identified the phosphorylation sites of TRADD which were induced by TNF and inhibited by TAK1 inhibitor 5Z7 using mass spectrometry. We found that TAK1 could mediate the phosphorylation of multiple sites in TRADD upon TNF stimulation. We next performed in vitro kinase assay using recombinant human TAK1 and human TRADD, followed by mass spectrometry to identify the sites directly phosphorylated by TAK1.