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The USP46 complex is a positive regulator of Wnt signaling. A–C Overexpression of the USP46 complex stimulates Wnt activation and stabilizes β-catenin levels. HEK293 STF cells were transfected and treated with recombinant Wnt3a (10 ng/ml) for 24 h as indicated. A Individual and B pairwise overexpression of USP46, FLAG-tagged WDR20, and FLAG-tagged UAF1 in HEK293 STF cells in the presence of Wnt3a leads to lower Wnt reporter (TOPFlash) activity compared to overexpression of all three members. The asterisk in (A) indicates a longer exposure for the USP46 blot. C Overexpression of the USP46 complex (Tri46) in HEK293 STF cells potentiates Wnt3a-mediated TOPFlash and promotes β-catenin stabilization. For transfections, the amount of total DNA was kept constant by the addition of vector plasmid. p values compare vector-transfected with USP46 complex-transfected cells. Tubulin is loading control. D, E Knockdown of USP46 and UAF1 by siRNA inhibits Wnt signaling and decreases β-catenin levels. HEK293 STF cells were transfected with non-targeting control (NT) or two independent (D) USP46 or (E) UAF1 siRNAs and treated with recombinant Wnt3a. Immunoblotting confirmed the knockdown of USP46 and UAF1 proteins. GAPDH is loading control. All graphs show mean ± SD of TOPFlash normalized to vector control in the presence of Wnt. p values compare NT treated to USP46 or UAF1 siRNA-treated cells. p ≥ 0.05 is not significant (ns). Significance was analyzed by one-way ANOVA followed by Tukey’s multiple comparisons test. Graphs all show a representative of n = 3 biologically independent experiments. All immunoblots are representative of at least three independent experiments. Source data are provided as a Source data file.
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