Fig. 5: Functional consequences of the BCA-associated FBXW11 p.F517S and β-catenin p.I35T substitutions.

a FBXW11^F517S and β-catenin^I35T are stable proteins. Western blot (WB) and quantitative analyses of V5-tagged FBXW11 and Flag-tagged β-catenin levels in transfected COS-1 cells, basally and after cycloheximide (CHX) treatment. GAPDH was the loading control. M, protein markers. Representative blots shown; data are expressed as mean ± SEM of three biological replicates. Two-way ANOVA followed by Sidak’s multiple comparison test was used to test significance (*, p = 0.047). b FBXW11^F517S has impaired binding to β-catenin. Lysates from transfected HEK293T cells expressing V5-tagged FBXW11^WT or FBXW11^F517S proteins and serum-starved were immunoprecipitated with anti-β-catenin antibody and assayed by WB (two biological replicates). c Lysates from transfected HEK293T cells co-expressing FBXW11^WT or FBXW11^F517S with Flag-tagged β-catenin and serum-starved were immunoprecipitated with anti-Flag or anti-V5 antibody and assayed by WB (two biological replicates). d β-catenin^I35T has impaired binding ability to FBXW11. Lysates from transfected HEK293T cells co-expressing β-catenin^WT or β-catenin^I35T with V5-tagged FBXW11^WT and serum-starved were immunoprecipitated with anti-Flag or anti-V5 antibody and assayed by WB (two biological replicates). e Relative protein levels and subcellular localisation of serum-starved COS-1 cells co-expressing V5-tagged FBXW11 and Flag-tagged β-catenin revealed by confocal microscopy analysis. Representative images from confocal maximum z-projections of cells co-expressing β-catenin^WT and FBXW11^WT (top panels), β-catenin^WT and FBXW11^F517S (middle panels), and β-catenin^I35T and FBXW11^WT (lower panels). Cells were stained with anti-Flag (green) and anti-V5 (magenta) antibodies. Merged images are shown in the right panels. Scale bar: 20 μm. Bar plots reporting the quantification of the green fluorescent intensity relative to β-catenin expression for the different experimental groups within the entire cell, nucleus or cytoplasm. For each cell, fluorescent intensity is normalised to the cell size (area). Data are expressed as mean ± SEM; n = 17 cells (β-catenin^WT and FBXW11^WT), n = 26 cells (β-catenin^WT and FBXW11^F517S), n = 24 cells (β-catenin^I35T and FBXW11^WT). Brown-Forsythe and Welch one-way ANOVA followed by Dunnett’s multiple comparison post hoc test was used to test statistical significance (*, p = 0.0131; **, p = 0.0014; * p = 0.0194). Source data are provided as a Source Data file.