Extended Data Figure 9: Characterization of A20 ZnF mutants and their cellular effects.

a, Summary of binding data of wild-type human A20 ZnF motifs and ZnF mutants to mono-ubiquitin, as measured by NMR, and to tri-ubiquitin chains, as measured by biolayer interferometry. Data are shown in Fig. 5b and Supplementary Information h, g. b, Analysis of TNF-engaged TNFR1 in untreated or TNF-treated E1A transformed A20 wild-type or A20 OTU(C103A) MEFs. Anti-TNF immunocomplexes were captured using anti-TNFR1 antibody-coupled beads and elutions were blotted for TNFR1. c, Analysis of TNFR1-associated RIPK1 K48 ubiquitination (Ub) status in TNF-treated wild-type and A20 ZnF4(C609A,C612A) MEFs. Immunoblot analysis of the corresponding whole-cell lysates are shown in the lower panels. d, Flag-wild-type A20 ubiquitinates recombinant murine TNFR1 with K48 chains. Flag–wild-type A20 or Flag–A20 ZnF4(C609A,C612A) proteins purified from HEK 293T lysates were added to in vitro reactions with recombinant murine TNFR1 and ubiquitin system enzymes. Reactions were immunoprecipitated in 4 M urea using an anti-K48 ubiquitin antibody, and immunoblotted, or reaction inputs were blotted as indicated. e, IκKβ-phosphorylated A20, but not IκKβ alone, promotes in vitro ubiquitination. For gel source data, see Supplementary Fig. 11. Data represent two to three biological replicates.