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. 2024 Jan 2;14:218. doi: 10.1038/s41598-023-50763-4

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© The Author(s) 2024

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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Detection of NICD heterodimer complexes on DNA. (A) 293T cells were transfected with combinations of FLAG-tagged N1ICD or N4ICD and FLAG- or HA-tagged binding NICD partners. ChIP was performed by two-step Co-IP with anti-FLAG then anti-HA antibodies. A positive control reaction was transfected with FLAG-tagged N1ICD alone and subjected to anti-FLAG Co-IP. A negative control reaction was transfected with HA-tagged N1ICD and subjected to anti-FLAG and anti-HA Co-IP. Hes1 and Hes4 promoter sequences were detected by PCR with antibodies flanking the head-to-head binding sites in each promoter. Shown is a representative image of a single experiment that was performed three independent times with similar results (B) 293T cells were transfected with combinations of FLAG-tagged N1ICD or N4ICD and FLAG- or HA-tagged binding NICD partners. ChIP was performed by two-step Co-IP with anti-FLAG then anti-HA antibodies. Positive control reactions (a and b) were transfected with FLAG-tagged N1ICD alone and subjected to anti-FLAG Co-IP. A negative control reaction (c) was transfected with HA-tagged N1ICD and subjected to anti-FLAG and anti-HA Co-IP. SPS 16 and SPS 21 promoter sequences were detected by PCR with antibodies flanking the head-to-head binding sites in each promoter. Shown is a representative image of a single experiment that was performed three independent times with similar results.