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. 2023 Nov 8;623(7988):803–813. doi: 10.1038/s41586-023-06717-x

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© The Author(s) 2023

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 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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Extended Data Fig. 3 — (a) Schematic representation of the alternative NF-κB pathway and the function of the p52/RelB and p52/p52 heterodimers (left panel); a graphical overview of the luciferase assay for testing the p52 function of the NFKB2 variants (middle panel); and a schematic representation of the functional consequences of the WT, p52^GOF and p52^LOF variants in the luciferase assay (right panel). (b) Relative luciferase activity (RLA) of WT or RelA-deficient HEK293T cells transfected with a κB reporter construct (κB-luc) in the absence or presence of plasmids encoding NIK and/or RelB for 24, 48 or 72 h. Results are expressed as the RLA normalized against the value for the EV. Bars represent the mean values (± s.d.) from 3 independent experiments performed in technical duplicates. (c) Western blot of HEK293T cells transfected for 24 h (left panel) or 48 h (right panel) in the absence or presence of plasmids encoding NIK and the WT or previously reported biochemical NF- κB2/p100 mutants. Data representative of two independent experiment are shown. (d) Luciferase assay testing the NF-κB2/p100 biochemical mutants (left) or deleterious variants from patients (right), 24 h after transfection. Bars represent the mean values (± s.d.) from more than 3 independent experiments.