β-catenin modified with atypical ubiquitin chains activates the LEF-TCF reporter. (A) Cell-based ubiquitination assays were carried out using HA-Ub mutants that contain only 1 intact lysine known to be involved in ubiquitin chain extension. The other 6 lysines in any given mutant HA-Ub are mutated from lysine to arginine and cannot participate in ubiquitin chain extension. We performed the ubiquitination assays using the V5-tagged K19R-K49R-β-catenin mutant and treated the cells with BIO (GSK3-β inhibitor). The ability of FANCL to catalyze atypical ubiquitin chain extension is compared with the ligase-inactive C307A-FANCL mutant. The experiment was carried out 2 independent times. (B) We performed LEF-TCF reporter assays to determine whether these ubiquitinated β-catenin subtypes are active. Cells were transfected with VC or FANCL, an HA-tagged ubiquitin as shown, and a LEF-TCF luciferase reporter. Luciferase activity was measured and shown are the results from 5 experiments normalized to the VC condition transfected with the knockout (KO) ubiquitin (mean ± SEM). The table below the figure is a compilation of all statistical analyses. Highlighted by shading are P values < .05. We compare FANCL (gray bars) versus vector-control (white bars) for the individual ubiquitin mutants (top row) and all ubiquitin mutants versus its own KO control for FANCL expression (within gray bars, middle row) and for vector-control (within white bars, bottom row). *P < .03 and highlights that the expression of lysine-11 ubiquitin activates the LEF-TCF reporter to the closest approximation of WT ubiquitin expression in FANCL-transfected cells. n/a indicates not applicable.