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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. 4 — (a) Western blot of HEK293T cells transfected for 48 h in the presence or absence of plasmids encoding NIK and WT or mutant NF-κB2, showing phosphorylated Y866-p100 (P-p100) levels, and p100 and p52 expression. Data representative of three independent experiments are shown. (b) Relative luciferase activity (RLA), indicating the p100/NF-κB2-dependent capacity to repress κB transcriptional activity for luciferase in HEK293T cells in the presence or absence of plasmids encoding NIK and various amounts of a plasmid encoding the C-terminal part of p100/NF-κB2 (Cter, aa 405-900) from WT or mutants, 48 h after transfection. The results are expressed as a percentage of the κB-luc RLA after transfection with NIK alone (left panel); the kinetic effect of transfection with NIK either alone or together with a plasmid encoding the various Cter constructs, as assessed by κB-luc transcriptional repression, from 24 to 72 h after transfection is shown in the right panel. Bars represent the mean values (± s.d.) from two independent experiments performed in duplicate. (c) Kinetic effect of transfection with NIK alone or together with a plasmid encoding the dimer-deficient Y247A single (p100^Y247A) or double (p100/^Y247A/W270*, p100^Y247A/R611*, p100^Y247A/S866N, or p100^Y247A/R853*) mutants, in terms of κB transcriptional repression of luciferase activity from 24 to 72 h after transfection. Results are expressed as a percentage of the κB RLA after transfection with NIK alone. Bars represent the mean values (± s.d.) from two independent experiments performed in duplicate. (d) Western blot of HEK293T cells cotransfected with a plasmid encoding NIK, together with various amounts of a plasmid encoding the WT or mutant NF-κB2 variants, together with a constant amount of empty vector (left panel) or of WT NFKB2 (right panel), showing phosphorylated p100 (P-p100), p100 and p52 expression. Data representative of three independent experiments are shown.