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. 2018 Feb 14;8:3019. doi: 10.1038/s41598-018-21329-6

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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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KLC1 plays a role regulating the sub-cellular distribution of CCDC28B. (A) Confocal microscopy analysis of endogenous CCDC28B (green) in hTERT-RPE cells showing localization of the protein in the cytoplasm, pericentriolar region/basal body (arrows illustrate examples in a ciliated and a non-ciliated cell, higher magnification in yellow box) and the nucleus (circle). Basal bodies and cilia axoneme were stained with anti-γ- and anti-acetylated α-tubulin respectively (red). (B) CCDC28B (green) signal is increased in the nucleus upon KLC1 KD (S.KLC1; lower panels) compared to control cells (S.CTRL; upper panels). In both (A,B), DAPI was used to stain nuclei. Scale bars correspond to 10 μm. (C) Sub-cellular fractionation assay using hTERT-RPE cells transfected with a Myc-CCDC28B expressing plasmid together with stealth control (S.CTRL) or stealth KLC1 (S.KLC1). CCDC28B is present in both the cytosolic and nuclear fractions in control cells and accumulates in the nuclear fraction in KLC1 KD cells. The membrane was cut at the 35 KDa ladder band. The blot incubated with the α-Myc to visualize CCDC28B was stripped and probed with α-Histone. The graph shows the nuclear/total (nuclear + cytoplasmic) ratio obtained by quantifying the western blot bands by densitometry. α-tubulin was used to normalize the nuclear intensity of CCDC28B and compensate for the cytosolic contamination in the nuclear fraction.