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. 2019 Oct 25;9:15296. doi: 10.1038/s41598-019-51773-x

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© The Author(s) 2019

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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TMX2 interacts with nuclear import protein. (A) Flag-TMX2 was overexpressed in HEK293 cells and immunoprecipitated with anti-Flag antibody. The band indicated by the arrow was analyzed by LC-MS. (B) Endogenous TMX2-intereacting proteins in HEK293 cells were immunoprecipitated with anti-TMX2 antibody and analyzed by LC-MS. (C) HEK293 cell lysates were immunoprecipitated with anti-TMX2 antibody and analyzed by western blot with anti-importin-β antibody. (D) mCherry-importin-β was expressed in HEK293 cells and immunostained with anti-TMX2 antibody. (E) Flag-Ran WT or -Ran Q69L mutant was overexpressed in cells and immunoprecipitated with anti-TMX2 antibody. (F) TMX2-VN was co-expressed with importin-β-VC, HA-RanWT-VC, or HA-RanQ69L-VC in HEK293 cells, and the overexpression of these proteins were confirmed by anti-TMX2, anti-VC, or anti-HA antibody. Venus fluorescence as BiFC signal indicates the interaction of these fusion proteins. BiFC signal was also detected by co-expression of Ran-VN and importin-β-VC as a positive control. (G) Co-localization of endogenous TMX2 and Flag-emerin was analyzed by immunofluorescence. (H) A diagram of TMX2 WT and isoform 2, which lacks part of the transmembrane region. (I) TMX2 WT and isoform 2 cDNA were amplified by PCR from HEK293 cDNA with the primer sets at 191–507th of TMX2 WT nucleotide. (J) Venus fused with TMX2 WT or isoform 2 was expressed in HEK293 cells, and co-localization with emerin-Flag was analyzed.