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. 2019 Sep 18;10(10):692. doi: 10.1038/s41419-019-1937-y

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 4 — a A20^+/+ and A20^−/− MEFs were stimulated with 1 µg/ml FLAG-hTNF for the indicated duration. TNFR1 Complex I was subsequently FLAG-immunoprecipitated. Protein levels were determined by immunoblotting. b A20^+/+ and A20^−/− HaCaTs were pretreated with CHX for 30 min before stimulation with 1 µg/ml FLAG-hTNF for the indicated duration. TNFR1 Complex I was subsequently FLAG-immunoprecipitated. Protein levels were determined by immunoblotting. c A20^+/+ and A20⁻^/− MEFs were stimulated with 1 µg/ml FLAG-hTNF for the indicated duration. TNFR1 Complex I was FLAG-immunoprecipitated, followed by USP2 + ƛ-phosphatase treatment on the post-IPs. Protein levels were determined by immunoblotting. d A20^−/− MEFs lentivirally reconstituted with wild-type A20, empty vector (EV) or indicated A20 mutants were stimulated with 1 µg/ml FLAG-hTNF for the indicated duration. TNFR1 Complex I was subsequently FLAG-immunoprecipitated. Protein levels were determined by immunoblotting. e A20^−/− MEFs lentivirally reconstituted with wild-type A20, empty vector (EV) or indicated A20 mutants were stimulated with 1 ng/ml mTNF and cell death was measured in function of time by SytoxGreen (SG) positivity. f A20^−/− MEFs lentivirally reconstituted with wild-type A20, empty vector (EV) or indicated A20 mutants were stimulated with 1 µg/ml FLAG-hTNF for the indicated duration. TNFR1 Complex I was subsequently FLAG-immunoprecipitated, followed by USP2 treatment on the post-IPs. Protein levels were determined by immunoblotting. Cell death experiments are presented as mean ± SEM of three independent experiments. Statistical significance for the cell death assays was determined using one-way ANOVA followed by a Dunnett post hoc test. Significance between samples is indicated in the figure as follows: *p < 0.05; **p < 0.01; ***p < 0.001; ns non significant.