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. 2024 May 7;43(12):2424–2452. doi: 10.1038/s44318-024-00104-6

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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/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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(A) HeLa cell lysates were subjected to pull-down with GST, GST-CENP-U (1–39), GST-CENP-U (1–50), or GST-CENP-U (1–60), followed by immunoblotting with antibodies for Scc1 and SA2, and CBB staining. (B) Multiple sequence alignment for the N-terminus of CENP-U. The DVFDF motif that is conserved in mammals are marked with *****. (C) Lysates prepared from HEK-293T cells transiently expressing CENP-U-GFP (human, chicken, and chimeric chicken with the DVFDF motif) were subjected to pull-down with GST or GST-Scc1 (281–420)-SA2 (80–1060), followed by immunoblotting with the antibody for GFP, and CBB staining. (D) Cartoon presentation of the overall structure of Scc1-SA2-CENP-U complex colored in purple, cyan, and orange, respectively (left), and the zoom view for binding details between F44 and F46 of CENP-U and Scc1-SA2 (right). (E) HeLa cell lysates were subjected to pull-down with GST or GST-CENP-U (1–60) in the forms of WT, ADA, FDA, and ADF, followed by immunoblotting with antibodies for Scc1, SMC1, SA2, and α-Tubulin, and CBB staining. Irrelevant lanes were removed. (F) The GST-Scc1 (281–420)-SA2 (80–1060) sub-complex was subjected to pull-down with MBP-H2A or MBP-CENP-U (1–200) in the forms of WT and ADA, followed by immunoblotting with the antibody for GST, and CBB staining. (G) Lysates prepared from HEK-293T cells transiently expressing CENP-U-GFP in the forms of WT and ADA were subjected to pull-down with GST or GST-Scc1 (281–420)-SA2 (80–1060), followed by immunoblotting with the antibody for GFP, and CBB staining. (H, I) U2OS-LacO cells transiently expressing the indicated proteins were stained with antibodies for the Flag-tag, Myc-tag, and DAPI. Example images are shown (H). The white arrows point to the LacO repeats. Scale bars, 10 µm. The fluorescence intensity ratio of SFB-CENP-U/EGFP at the LacO repeats was quantified in 30 cells for each condition, with statistics being performed using one-way ANOVA (I). Means and SDs are shown. NS no significance. Source data are available online for this figure.