Characterizing the regions that are required for direct interaction between FANCB and FANCL. (A) Mammalian 2-hybrid assays coexpressing mutant fragments of BD-FANCL with full-length AD-FANCB. Fold induction of luciferase expression was measured relative to AD-FANCB alone. +++ indicates a strong activation of the luciferase reporter gene; –, no activation of the reporter gene. (B) FANCL constructs that failed to activate the luciferase reporter in the mammalian 2-hybrid experiments were transfected in 293 cells. Whole-cell extracts were immunoblotted with anti–GAL4-BD to show expression of the constructs. The fragments 219-375 and 275-375 are probably difficult to detect because of the presence of background bands. (C) BD-FANCL–containing point mutations of the RING domain were coexpressed with full-length AD-FANCB and fold induction was measured relative to AD-FANCB alone. +++ indicates a strong activation of the luciferase reporter gene; –, no activation of the reporter gene. (D) FANCL missense mutants were transfected in FA-L cell line EUFA868 and tested for their ability to restore the MMC hypersensitive phenotype of this cell line in an MMC growth inhibition test.