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. 2022 Apr 14;29(4):403–413. doi: 10.1038/s41594-022-00758-y

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© The Author(s) 2022

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. 4 — a, CENP-A distribution in sucrose gradient in the presence of WT CENP-N (gray) and after IAA-induced degradation of CENP-N (orange). An equal amount of chromatin was loaded per well on an SDS gel, resolved by blotting against H4 histone (Extended Data Fig. 9l). b,c, Comparison of CENP-A distribution in the presence of transiently expressed CENP-N variants, WT CENP-N (gray), K102A CENP-N (lilac), and 7-ala CENP-N (pink, 7 positively charged amino acids on α6 are mutated to alanine). Dots in a–c represent a mean of two biological replicates. I_max and I_i in a–c represent a maximum signal intensity from all fractions and the signal intensity in a given fraction, respectively. Solid lines represent interpolation between experimental data points (using Akima spline). d, Representative images of nuclei (Hoechst stain) showing endogenous WT CENP-N (GFP) or transgenic CENP-N variant (anti-Ruby antibody) localization at centromeres (CREST signal). Scale bar, 5 µm. Insets show magnified images of example centromeres (CREST foci). Cells were treated for 24 hours with doxycycline (dox) to induce expression of mRuby2-3×FLAG-tagged CENP-N (WT or mutant) and/or with indole acetic acid (IAA) to deplete endogenous auxin-inducible degron (AID)-tagged CENP-N-GFP as indicated. e,f, Normalized centromeric fluorescence signal corresponding to endogenous WT CENP-N (GFP) (e) or transgenic WT or CENP-N variant (anti-mRuby antibody) (f). Each dot represents the median centromeric CENP-N signal from >100 cells from 1 biological replicate. Line and error bars are mean of three biological replicates ± s.d.

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