Effect of FANCB variants on ubiquitination activity of the FA core complex. (A) Schematic of in vitro ubiquitination reaction. (B) Results of an example monoubiquitination assay; Coomassie-stained sodium dodecyl sulfate–polyacrylamide gel electrophoresis gel to compare WT BL100 with BL100 with FANCB variants in 90-minute ubiquitination reaction. Western blotting of FANCD2 and FANCI reveals the extent of ubiquitinated protein, calculated from western blots using anti-StrepII-Fancd2 (green) or anti-Flag-FancI (red) (FANCI). Two different preparations of L717* complex are shown. (C) Example of time course experiment comparing monoubiquitination activity of WT and L717*-FANCB containing reactions by western blot (above) or quantified ubiquitinated forms of FANCD2 and FANCI. Similar results were obtained from n = 4 experiments.