FANCL ubiquitinates β-catenin. (A) 293FT cells were transfected with a maxGFP control (asterisk), C307A-FANCL mutant (Mu), or WT FANCL (WT); V5-tagged WT, K19R, K49R, or K19R-K49R β-catenin; and hemagglutinin-tagged ubiquitin (HA-Ub). The C307A-FANCL is a ligase-inactive mutant. The β-catenin mutants were used to diminish the contribution of the GSK3-mediated destruction complex targeting β-catenin for polyubiquitination. Whole cell extracts were subjected to immunoprecipitation with V5 antibody and the blots were probed with HA antibody. This experiment was performed 2 independent times for each set. The exposure times for these blots are indicated for comparison of relative signal with or without BIO (GSK3-β inhibitor). We quantified ubiquitinated β-catenin by performing densitometry analysis using ImageJ Version 1.34u between the 98 kDa and 148 kD markers for each blot and expressed the relative signal to the maxGFP control. We also probed the same blots for total β-catenin to show relatively equal immunoprecipitation of total β-catenin. (B) LEF-TCF reporter assays using luciferase as the readout. For this experiment we used 2 ligase-inactive FANCL mutant controls. Raw luciferase values were normalized to FANCL expression relative to C307A-FANCL expression. The results shown are from 3 independent experiments (mean ± SEM); P values are < .01 for the differences noted. (C) Estimation of β-catenin turnover was determined in [³⁵S]-methionine and cysteine pulse-chase labeling experiments to compare the effect of control versus FANCL overexpression. Shown is a representative blot from 3 experiments. The graph summarizes relative β-catenin level to time zero of the “chase,” which begins when the labeling media is replaced with complete unlabeled media (P values are indicated on the graph).