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. 2018 Sep 6;9:3613. doi: 10.1038/s41467-018-05808-y

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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. 3 — Nestin contains a nuclear localization signal (NLS). a Confocal images of Nestin immunostaining (green) in A549 and H1299 cells. Scale bars, 5 μm. b Immunoblotting for subcellular fractions of Nestin in A549 and H1299 cells (T total cell lysates, C cytoplasmic cell lysates, N nuclear cell lysates). c Immunohistochemical staining of Nestin in A549 and H1299 cells. Scale bars, 30 μm. d Upper: three putative NLSs identified by bioinformatic analysis. Lower: schematics of the Nestin constructs used for the localization study. Each fusion protein was expressed with a GFP tag (left), and its localization was assessed (right; C, predominantly cytoplasmic; C + N, in both compartments; PE, at the periphery of outer nuclear envelope). e Confocal images of GFP-Nestin fusion protein in A549 cells. Nuclei were marked by DAPI (blue). Scale bars, 20 μm. f Immunoblotting analysis of GFP-Nestin distribution in A549 cells (left; C cytoplasmic cell lysates, N nuclear cell lysates) and the corresponding cytoplasm/nucleus intensity ratio (CN ratio) of GFP-tagged proteins (right). g, h Schematics of the GFP-tagged Nestin constructs showing the sequences (g) and positions (h) of a potential NLS and the targeted aa substitutions (asterisk) used to disable it. Scale bars, 10 μm