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. 2023 Oct 5;14:6173. doi: 10.1038/s41467-023-41836-z

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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. 5 — A, B The catalytic activity of USP46 is required for its regulation of LRP6 levels. Cells expressing the wild-type or mutant USP46 complexes were incubated in the absence or presence of Wnt3a, and immunoblotting was performed. A Overexpression of the USP46 complex containing the catalytically dead USP46^C44S (Tri46^C44S) does not increase LRP6 levels. B Overexpression of the USP46 complex containing the USP46 binding mutant, FLAG-UAF1^S170Y, does not increase LRP6 levels. GAPDH is loading control. C–E His-ubiquitylation assays. HEK293 cells were transfected as indicated, lysed under denaturing conditions, and His-Ub (hexahistidine-tagged ubiquitin) modified proteins isolated by nickel affinity purification. LRP6 and FLAG-tagged proteins were detected by immunoblotting with anti-LRP6 and anti-Flag antibodies, respectively. C Overexpression of FLAG-RNF43 promotes endogenous ubiquitylation of LRP6, which is opposed by overexpression of the USP46 complex. D Overexpression of FLAG-RNF43 in LF203 cells similarly promotes LRP6-FLAG ubiquitylation, which is opposed by overexpression of the USP46 complex. E Knockdown of USP46 enhances LRP6-FLAG ubiquitination. WCL = whole cell lysates. F The sensitivity of intestinal organoids to USP46 depletion is RSPO-dependent. Exogenous RSPO is essential for culturing intestinal organoids in vitro. Intestinal organoids infected with a control lentivirus or two independent lentiviruses expressing USP46 shRNAs were grown with decreasing RSPO conditioned media (CM). Viability was assessed by Cell-Titer Glo. p ≥ 0.05 is not significant (ns). Significance was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Graphs show mean ± SD (n = 3 independent experiments). GAPDH is loading control. All immunoblots are representative of at least three independent experiments. Source data are provided as a Source data file.