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The USP46 complex acts at the level of the Wnt receptor, LRP6. A, B USP46 is upstream of the β-catenin destruction complex. A USP46 depletion does not block the stabilization of β-catenin upon AXIN1 siRNA knockdown. HEK293 cells were transfected with USP46 and AXIN1 siRNAs as indicated and immunoblotted for β-catenin. Immunoblotting for AXIN1 and USP46 confirmed their knockdown. B The tankyrase inhibitor, XAV939, which stabilizes AXIN to promote β-catenin degradation, inhibits β-catenin stabilization mediated by the USP46 complex. Cells were transfected and treated with Wnt3a in the absence or presence of XAV939 (1 μM) as indicated and immunoblotted for β-catenin, USP46, FLAG-UAF1, and FLAG-WDR20. C, D Overexpression and knockdown of the USP46 complex increases and decreases steady-state levels of LRP6, respectively. Cells were transfected as indicated, incubated in the absence or presence of Wnt3a, and immunoblotted for LRP6. C HEK293 cells were transfected with the USP46 complex (Tri46) in the presence of Wnt3a. D HEK293 cells were transfected with non-targeting (NT) or USP46 siRNAs. Graph shows mean ± SD of TOPFlash normalized to non-targeting control in the presence of Wnt. p-values compare cells incubated with NT versus USP46 siRNA in the presence of Wnt3a. Significance was analyzed by two-tailed Student’s t-test. The graph shows a representative of n = 3 biologically independent experiments. E Wnt signaling promotes the association between the USP46 complex and endogenous LRP6. HEK293 cells were transfected with USP46 complex components and treated with Wnt3a as indicated, FLAG-UAF1 (*) and FLAG-WDR20 (**) immunoprecipitated (IP) with anti-FLAG conjugated beads, and co-immunoprecipitated LRP6 detected by immunoblotting. WCL whole cell lysates. F The USP46 complex increases cell-surface levels of LRP6. HEK293 cells were transfected with USP46 complex components and treated with Wnt3a, as indicated. Cells were then surface biotinylated, lysates subjected to neutravidin-pulldown, and immunoblotted for endogenous LRP6 and insulin (IR, control) receptors. WCL whole cell lysates. Tubulin and GAPDH are loading controls. All immunoblots are representative of at least three independent experiments. Source data are provided as a Source data file.
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