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. 2017 Oct 20;7:13690. doi: 10.1038/s41598-017-13989-7

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© The Author(s) 2017

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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Characterization of the minimal regions of Notch4/Int3 required for intermolecular activation of NF-κB. (A) To map the region(s) of NICD4/Int3 that mediate interaction with NF-κB, we generated a series of Int3 deletion constructs. The regions of Int3 that remain in each deletion mutant is indicated. The numbers above the maps indicate an amino acid residue at indicated boundaries. (B) In vitro translation products of the Int3 deletions. RAM (lane 1), ANK (lane 2), RAM-ANK (lane 3), PB-PEST (lane 4) and full length Int3 (lane 5). (C) NF-κB reporter assay of HC11, HC11-Int3, HC11-RAM, HC11-ANK, HC11-RAM-ANK, HC11-PB-PEST cells. The ANK repeats region of Int3 caused the highest activation of NF-κB reporter. **P < 0.01 compared HC11 cells. (D) HC11 cells were transfected with expression vectors expressing the Int3, RAM, ANK, RAM-ANK, and PB-PEST deletion mutants compared to HC11 and HC11-Int3 cells for their ability to confer on HC11 cells the capability for anchorage independent in soft agar. (E) HC11 cells expressing Int3, RAM, ANK, RAM-ANK and PB-PEST deletion mutants compared to HC11 and HC11-Int3 cells in an invasion assay (see Materials and Methods). In panel (C–E), each bar represents the mean ± SEM of a minimum of a duplicate of three independent experiments for each experimental group.