Extended Data Figure 6: In vitro deubiquitination assays.
From: Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation
Additional data corresponding to Fig. 2. Normalized A20 WT or OTU(C103A) inputs purified from E. coli or mammalian HEK 293T cells are shown in Fig. 2a. a, A20-mediated cleavage efficacy of K63- or K48-linked tetraubiquitin conjugated to a HA-tagged RIPK1 peptide. b, Sequence of the HA epitope-tagged human RIPK1 peptide. The HA epitope tag is shown in blue, the human RIPK1 residues in black, and K377 is highlighted in red. c, Cleavage time course of linear tetraubiquitin by purified A20 WT or OTU(C103A) from E. coli or from mammalian HEK 293T cells. Input protein levels are shown in Fig. 2a. d, A schematic of the human A20 protein indicating where the phosphorylation sites are localized. Mass spectrometry PhosphoSite analysis (http://www.phosphosite.org/) of A20 derived from mammalian expression systems is shown in Supplementary Information d. e, Comparison of the cleavage efficacy of K63-linked tetraubiquitin with increasing doses of human wild-type A20 or phospho-site mutant A20 (4× phos mut). 4× phos mut: S381A, S480A, S565A, and T625A. Wild-type or phos mut A20 proteins were expressed in and purified from mammalian HEK 293T cells. f, Tandem mass spectrum for the S381-containing peptide from human A20 expressed in E. coli and phosphorylated with recombinant IκKβ. g, Cleavage efficacy of linear tetraubiquitin chains by increasing doses of E. coli-derived wild-type A20, IκKβ alone, or IκKβ-phosphorylated A20. For gel source data, see Supplementary Figs 8, 9. Data represent two to five biological replicates.
