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. 2023 Oct 16;42(47):3514–3528. doi: 10.1038/s41388-023-02858-5

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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 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. 4 — A The flow chart for identification of KLHL29-interacting proteins. BT549 and CAL51 cells stably expressing pCDH-Flag or pCDH-Flag-KLHL29 were subjected to IP assays, followed by MS analysis. B The Venn diagram analysis of KLHL29-interacting proteins. 46 proteins interact with KLHL29 in BT549 cells, and 69 proteins interact with KLHL29 in CAL51 cells. C The list of eight potential KLHL29-interacting proteins in both BT549 and CAL51 cells. D, E Exogenous KLHL29 interacts with exogenous DDX3X. HEK293T cells were transfected with plasmids encoding Flag-KLHL29 and Myc-DDX3X. Whole-cell lysates were immunoprecipitated with an anti-Flag or anti-Myc antibody and subjected to western blotting analysis with indicated antibodies. F Schematic diagram of the different KLHL29 truncation constructs used. G DDX3X interacts with the Kelch domain of KLHL29. HEK293T cells were transfected with the plasmids encoding the Flag-KLHL29 fragments along with the Myc-DDX3X plasmid, followed by a set of co-IP and western blotting assays. H Immunofluorescent images show the co-localization of Flag-KLHL29 (green) and Myc-DDX3X (red) in BT549 and CAL51 cells. The nuclear was counterstained with DAPI (blue). I Overexpression of KLHL29 reduces DDX3X protein levels in BT549 (left), CAL51 (middle) and HEK293T (right) cells. J Knockdown of KLHL29 elevates DDX3X protein levels in MDA-MB-231 (left), SUM159PT (middle) and HEK293T (right) cells. K, L Immunohistochemical staining analysis of KLHL29 and DDX3X expression in TNBC specimens. Representative images are shown. M Negative correlation between KLHL29 and DDX3X expression levels in TNBC specimens. Correlation coefficients were performed using the Spearman test. Two-tailed P-values were calculated.