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. 2022 Jan 8;30(1):43–57. doi: 10.1007/s10577-021-09678-x

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© The Author(s) 2021

Open AccessThis 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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Fig. 2 — FRAP analyses of CENP-C-GFP and mScarlet-CENP-T during mitosis. (A) FRAP analysis of each cell line during mitosis evaluated using the indicated fluorescent proteins. DNA was stained with NucSpot™ Live 650 Nuclear Stain. Left panels are images before bleaching (pre-bleach). The boxed area was bleached, and signal recovery is shown at the indicated time points. The black scale bar indicates 10 μm. The white scale bar indicates 2.5 μm for magnified panels. (B) Solid lines display the means of quantitative FRAP measurements for CENP-C-GFP from at least 10 mitotic cells from each indicated cell line. The colored area between the two dashed lines indicates the standard error for each time point. (C) FRAP results for mScarlet-CENP-T in 10 mitotic cells from each indicated cell line. (D) I_max is the maximum recovery rate, t_1/2 is the half-time of recovery, and k_off is the recovery constant. These values were calculated through curve fitting. The residence time at the kinetochores was calculated as the inverse of the k_off value. The k_off and residence time at kinetochores for CENP-C^WT-GFP, CENP-C^Δ648-676-GFP, and CENP-C^T651A-GFP during mitosis and interphase are shown