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. 2020 Jun 19;11(12):894–914. doi: 10.1007/s13238-020-00734-6

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© The Author(s) 2020

Open AccessThis 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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/.

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TRIM35 promotes K63-linked polyubiquitination of TRAF3. (A) IB analysis of HEK293T cells expressing TRAF3-Flag and increasing amounts of TRIM35-V5. (B) Co-IP and IB analysis to assess TRAF3 ubiquitination in HEK293T cells expressing TRAF3 (C68A/H70A)-Flag, HA-ubiquitin, with or without V5-tagged TRIM35 or its DelRING mutant. (C) Co-IP and IB analysis to assess TRAF3 ubiquitination in HEK293T cells expressing TRAF3 (C68A/H70A)-Flag, with or without TRIM35-V5. (D) Co-IP and IB analysis to assess endogenous TRAF3 ubiquitination in stable TRIM35-overexpressing or control THP-1 cells infected with SeV at the indicated timepoints. (E) Co-IP and IB analysis to assess TRAF3 ubiquitination in HEK293T cells expressing TRAF3-Flag, with or without TRIM35-V5, HA-ubiquitin (K63), and HA-ubiquitin (K63R). (F) Mutants of human TRIM35 in which different cysteine residues were replaced. (G and H) Co-IP and IB analysis to assess TRAF3 ubiquitination in HEK293T cells expressing TRAF3-Flag, HA-ubiquitin, and V5-tagged TRIM35 or its mutants as in (F). (I) Co-IP and IB analysis of HEK293T cells expressing TRAF3-Myc, IRF3-Flag, along with increasing amounts of TRIM35-V5. (J) Co-IP and IB analysis of HEK293T cells expressing IKKε-Flag, with or without TRIM35-V5, followed by infection with SeV for 12 h. (K) Co-IP and IB analysis of HEK293T cells expressing IKKε-Flag, VISA-Myc, with or without TRIM35-V5. Data are representative of at least three independent experiments