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. 2021 Mar 2;12:1379. doi: 10.1038/s41467-021-21711-5

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© The Author(s) 2021

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. 3 — a Immunoprecipitation and immunoblot analysis of the interaction between hemagglutinin (HA)-tagged N4BP1 and Flag-tagged components of the NF-κB pathway in HEK293T cells transfected to overexpress HA-tagged N4BP1 (HA-N4BP1) alone (−) or together with vector encoding Flag-tagged NF-κB pathway components (above lanes). b Detection of the endogenous N4BP1-NEMO association in wild-type and N4bp1^−/− peritoneal macrophages stimulated with Pam3CSK4 (+) or left unstimulated (−), assessed by immunoprecipitation with rabbit IgG as a control, or with anti-NEMO, followed by immunoblot analysis with anti-N4BP1 or anti-NEMO. c, d Mapping the binding regions between HA-N4BP1 and Flag-NEMO. c Left, Schematic representation of full-length N4BP1 (F.L.) or N4BP1 deletion mutants and amount of NEMO they bind. Right, immunoprecipitation and immunoblot analysis of the interaction between Flag-NEMO and HA-N4BP1 deletion mutants. d Above, schematic representation of NEMO deletion mutants. Below, immunoprecipitation and immunoblot analysis of the interaction between HA-N4BP1 and Flag-NEMO deletion mutants. e Above, NF-κB-dependent luciferase reporter activity in wild-type or N4BP1^−/− HEK293T cells transfected with empty vector (−) or vectors encoding the indicated N4BP1 deletion mutants and stimulated with IL-1β or left unstimulated (none). Data points represent independent cultures (n = 3 independent transfections of three cultures split from one cell line). Below, immunoblot analysis of HA-N4BP1 or truncation mutants in the pooled, analyzed cells. FL full-length N4BP1. **P = 0.0065, ****P < 0.0001, ****P < 0.0001, **P = 0.005, *P = 0.0303, ***P = 0.0002, ***P = 0.0001, ****P < 0.0001, unpaired, two-tailed Student’s t test. Data are representative of two independent experiments (a–e). Mean ± s.d. plotted in e. P values are listed in order from left to right. Source data are provided in the Source Data file.