Fig. 5

Fig. 5 The USP46 complex deubiquitinates LRP6 and opposes the action of the Wnt receptor E3 ligase, RNF43.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.