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. 2018 Oct 22;9:4381. doi: 10.1038/s41467-018-06756-3

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© The Author(s) 2018

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. 5 — CYPJ disrupts the K63-linked ubiquitin-chain sensing of TAB2 and TAB3. a 293T cells were transfected with plasmids as indicated for 24 h. The enforced expressed Flag-TAB2 was immunoprecipitated with an anti-Flag antibody, and the polyubiquitin signals were detected with an anti-HA antibody. b WT or CYPJ-knockout (KO-1 and KO-2) 293T cells were transfected with or without HA-K63-Ub and Flag-TAB2 for 24 h, followed by IP and IB as indicated. c Lysates of WT or CYPJ-knockout 293T cells treated with TNF for different times were immunoprecipitated with an anti-TAB2 antibody, followed by IB with the indicated antibodies, including anti-K63 Ub. d 293T cells were co-transfected with Flag-TAB2-NZF, HA-K63-Ub, and Myc-CYPJ as indicated, and cell lysates were immunoprecipitated with an anti-Flag antibody followed by IB with an anti-HA antibody. e Lysates of Flag-TAB2 with or without HA-CYPJ-transfected 293T cells were incubated with in vitro synthesized His-K63-Ub chain (1 μg) as indicated. The His-K63-chain was immunoprecipitated with an anti-His antibody, and the bound Flag-TAB2 was detected with an anti-Flag antibody. f, g Binding of GST, GST-CYPJ, or GST-TAB2-NZF (f) or GST-TAB3-NZF (g) to the His-K63-chain was monitored by GST pull-down assays as indicated. Approximately 1 μg of the His-K63-chain was used in each reaction