Extended Data Figure 5: Additional analysis of ubiquitination status analysis of TNFR1 and associated proteins.

a, A summary table of selected proteins identified in anti-Flag immunocomplexes from untreated or in Flag–TNF-treated wild-type MEFs by LC-MS/MS analysis (left columns) and a summary of the ubiquitination sites identified on the indicated proteins from TNF-treated A20 wild-type MEFs with the PTMscan approach using anti-K-ε-GG antibodies and LC-MS/MS (right column). b, Analysis of TNF-engaged TNFR1 in untreated or Flag–TNF-treated E1A-transformed A20 wild-type or A20 OTU(C103A) MEFs, or in A20 null primary MEFs. Anti-Flag immunocomplexes were purified using Flag peptide elution and elutions were blotted for TNFR1. Immunoblots of the corresponding whole-cell lysates are indicated below. c, Lysates corresponding with Fig. 1c. d, Murine TNFR1(K256R) attenuates TNFR1 ubiquitination and downstream signalling. Murine wild-type or TNFR1(K256R) was transfected in human 293T cells and cells were treated with Flag–TNF as indicated. Equal inputs of lysates were immunoprecipitated with anti-Flag, dissociated and re-immunoprecipitated with anti-linear ubiquitin antibody, and blotted for murine TNFR1, or lysates were blotted with the indicated antibodies. e, Analysis of TNFR1-associated RIPK1 K63 ubiquitination (Ub) status and TNFR1 immunoprecipitates in Flag–TNF-treated wild-type and A20 OTU(C103A) MEFs. f, Comparison of activated TNFR1 ubiquitination status in E1A transformed A20 wild-type and OTU(C103A) MEFs. Treated cells were lysed in buffer containing 6 M urea and immunoprecipitated with the indicated antibodies under denaturing conditions. g, Summary table of RIPK1 ubiquitination sites identified from TNF-treated A20 wild-type and OTU(C103A) MEFs with the PTMscan approach using anti-K-ε-GG antibodies and LC-MS/MS. Peptides were quantified with area under curve (AUC) and summarized to site level. The equivalent human RIPK1 residues are also indicated. The average ratio of endogenous RIPK1 ubiquitination sites in A20 OTU(C103A):wild-type A20 is 1.7. Additional TNFR1 mass spectrometry data are shown in Supplementary Information a–c. For gel source data, see Supplementary Figs 7, 8. Data represent two to four biological replicates.