(A) Residue S80 (yellow spheres) is located at the centre of the Box:RING interface and is part of an epitope related to the IKKβ phosphorylation motif found in TRIF. (B) MS/MS spectra of TRIM21-His purified from 293 T cells indicating the presence of phosphorylated S80 (pS80) within a tryptic peptide precursor/m/z/680.0^3 ± 0.39 ppm. (C) ELISA of pS80 antisera at 1:50000 against recombinant WT or pS80 TRIM21. (D) IKKβ phosphorylates C-terminally His-tagged WT but not S80A T21-His, as detected by specific pS80 polyclonal sera. (E) As D except in presence of 30 nM bortezomib (Btzb) or DMSO and, below, NFκB stimulation by IKKβ in parallel. (F–H) In vitro phosphorylation of T21 RB by IKKβ with increasing ATP (F), impaired phosphorylation of S80A and I79R RB (G), in presence of 20 µM IKKβ inhibitor TPCA-1 (H). (I) In vitro IKKβ phosphorylation of full-length lipoyl-T21. (J) Phosphorylation of WT or S80A T21-His from control 293T (spCas9) or IKKβ-deleted 293T (spCas9/sgIKKβ) cells.