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. 2017 Nov 10;7:15311. doi: 10.1038/s41598-017-15497-0

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© The Author(s) 2017

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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Overexpression of NPRL2 promotes interaction with AIF and induces AIF translocation to the nucleus. (a) NPRL2 interacts with AIF. Immunoprecipitates were prepared from HEK293 cells stably expressing GFP or FLAG-NPRL2-GFP and analyzed by immunoblotting for the indicated proteins. (b) The scheme of subcellular fractionation experiment used for the detection of NPRL2 localization. (c) Overexpressed NPRL2 localizes to mitochondria. The subcellular localization of NPRL2 was detected by immunoblotting with antibodies against indicated proteins in the mitochondrial and cytosolic extracts. TOM20 was used as mitochondrial marker and GAPDH as cytosolic marker. (d–f) Overexpression of the NPRL2 induces re-localization of NPRL2 and AIF to the nucleus. The cytoplasm and nuclear extracts were prepared from HEK293 cells either stably expressing FLAG-NPRL2-GFP or GFP (d) or transfected with pFLAG or pFLAG-NPRL2 plasmids (e) and analyzed by immunoblotting for the indicated proteins with Histone H3 as a marker for the nucleus and GAPDH as a marker for the cytoplasm. (f) Immunofluorescence analysis of localization of NPRL2 and AIF localization. Nucleus is stained with DAPI (blue); NPRL2 (green) is detected either via GFP in case of HEK293 stably expressing FLAG-NPRL2-GFP or with immunofluorescence with anti-NPRL2 antibody; AIF (red) is detected with anti-AIF antibody. Scale bar, 5 μm. The increased level of NPRL2 expression in these experiments is indicated with growing green bar to the right of image panels.