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. 2018 May 14;9:1888. doi: 10.1038/s41467-018-04089-9

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© The Author(s) 2018

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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Fig. 1 — Serine 7-phosphorylated CENP-A (p-CENP-AS7) localizes to the inner side of centromeres during mitosis. a Immunofluorescence on metaphase chromosome spreads from HeLa-S3 cells, double-stained with anti-CENP-A and anti-p-CENP-AS7 antibodies. Merged image with DAPI staining (blue), scale bar = 10 µm. Magnification × 7.5 of a single chromosome. b Distribution of p-CENP-AS7 (green) and CENP-A (red) at centromeres. Left panels: different patterns of immunofluorescence signals with p-CENP-AS7 on the inner side only, on both the inner and outer sides and on the outer side only; right panels: 3D views of p-CENP-AS7 (green) and CENP-A (red) signals. Scale bar = 1 µm. In total, 520 chromosomes from 3 independent experiments were scored and the percentages of chromosomes with p-CENP-AS7 on the inner and/or outer side of the centromere are indicated on the right. c CENP-A and p-CENP-AS7 localize to different domains on mitotic stretched chromatin fibers. Extended chromatin fibers were prepared from HeLa-S3 cells blocked in mitosis by overnight nocodazole treatment; CENP-A (red) and p-CENP-AS7 (green) signals on a representative centromere chromatin fiber are shown. The lower panel shows the profile of signals along the length of the fiber. Scale bar = 5 µm. d Only a small amount of CENP-A displays serine 7 phosphorylation during mitosis. HeLa cell lines stably expressing CENP-A-WT-HA and CENP-A-S7A-HA were left untreated (left panel) or were treated overnight with nocodazole (right panel). Protein extracts were separated on a 20% polyacrylamide (29:1 acrylamide/bisacylamide) gel and immunoblotted with an anti-HA antibody. The shift due to phosphorylation is indicated by an asterisk. Three independent experiments were performed