Extended Data Figure 7: Effects of linear ubiquitination in modulating TNFR1 signalling and cell viability.

a, HOIP RNAi decreases linear ubiquitination of TNFR1. Wild-type MEFs were transfected with control or HOIP RNAi oligonucleotides and treated for the indicated times with TNF. Treated cells were lysed in buffer containing 6 M urea and immunoprecipitated with the indicated antibodies under denaturing conditions. Immunoprecipitates and whole-cell lysates were blotted as indicated. b, Area under the curve (AUC) data corresponding to cell death data in Fig. 3a. All error bars are s.e.m. for technical triplicates. ***P < 0.001 determined by t-test. c, Murine TNFR1(K256R) enhances caspase activation. Human HEK 293T cells were treated with control or with human TNFR1 RNAi oligonucleotides and transfected with murine wild-type or with TNFR1(K256R) as indicated. Cells were treated with TNF as indicated and equal inputs of lysates were immunoblotted with the indicated antibodies. d, Murine TNFR1(K256R) does not modulate MAPK or NF-κB signalling. Human HEK 293T cells were treated with control or with human TNFR1 RNAi oligonucleotides and transfected with murine wild-type or with TNFR1(K256R) as indicated. Cells were treated with TNF as indicated and equal inputs of lysates were immunoblotted with the indicated antibodies. e, Evaluation of the specificity of anti-caspase 8 antibodies. E1A-transformed MEFs of the indicated genotype were lysed in buffer containing 6 M urea, quantified, and immunoblotted with the indicated antibodies as detailed in the Methods. f, A summary table of cell death proteins identified in anti-Flag immunocomplexes from untreated or in Flag–TNF-treated wild-type MEFs by LC-MS/MS analysis (also see Supplementary Fig. 6a). g, Sequences of the caspase 8 peptides in anti-Flag immunocomplexes from untreated or in Flag–TNF-treated wild-type MEFs by LC-MS/MS analysis. h, Left panels, analysis of FADD immunoprecipitates and FADD-associated RIPK1 K63 ubiquitination (Ub) status in TNF-treated wild-type and A20 OTU(C103A) MEFs transfected with control- or HOIP RNAi oligonucleotides. Right panels, immunoblot analysis of whole-cell lysates from TNF-treated wild-type and A20 OTU(C103A) MEFs transfected with control- or HOIP RNAi oligonucleotides. HOIP knockdown was validated by RT–PCR analysis (data not shown). For gel source data, see Supplementary Figs 9, 10. Data represent two to three biological replicates.