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. 2023 Sep 6;14:5451. doi: 10.1038/s41467-023-41061-8

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© The Author(s) 2023

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 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. 7 — a Schematic showing steps for identifying HDCA-binding proteins using microarrays fabricated with recombinant human proteins. Created with BioRender.com. b Magnified image of Bio-HDCA binding to RAN, CRM1, PPARα spots on the protein array. Z score and I mean ratio are shown. c Representative western blots showing thermostable individual proteins following indicated heat shocks in the presence (+) or absence (−) of HDCA (100 μM) by CETSA. Quantification of thermostable RAN from CETSA (n = 3 biologically independent experiments). d Schematic showing interaction of HDCA with RAN/CRM1/PPARα export complex. e Co-IP assay of the association of CRM1/RAN and CRM1/PPARα in the presence of 100 μM HDCA in AML12 cells. f Co-IP assay of the association of Flag-GFP-RAN and HA-GFP-PPARα, Flag-GFP-RAN and GFP-CRM1, HA-GFP-PPARα and GFP-CRM1 in the presence of 100 μM HDCA in HEK293T cells. g Representative PLA images and duolink spot quantitation of PPARα/RAN, CRM1/RAN and PPARα/CRM1 interactions in AML12 cells (Scale bar, 25 μm). Representative images are shown for each condition from one of 3 biologically independent experiments. The graph shows the average number of PLA signals per cell, n = 4–8 random fields of view (42–60 cells of each condition). h Molecular docking between HDCA and RAN (RAN protein (gray), HDCA (blue), GLY-5 (green), PRO-49 (purple) and VAL-51 (pink)). i Co-IP assay of the association of Flag-GFP-RAN (G5A/P49A/V51A) and GFP-CRM1 in the presence of 100 μM HDCA (Gly-5, Pro-49, Val-51 of RAN were all mutated to Ala). Experiments in e, f, and i were repeated independently at least twice with similar results. Data are presented as mean values ± SEM. Differences between groups were determined by unpaired two-tailed Student’s t test. Source data are provided as a Source Data file.