FANCI is mislocalized in FA-I F010191 lymphoblasts. (A) Schematic representation of WT FANCI and mutant FANCI proteins expressed in F010191 FA-I cells. In silico predicted leucine zipper, armadillo (ARM) repeat domain, EDGE motif and NLS, and lysine 523 (the monoubiquitination target of the FA core complex). (B) Time course of FANCI and FANCD2 monoubiquitination after 2mM HU treatment in normal or F010191 LCLs evaluated by Western blot analysis with the indicated antibodies. Note the slightly faster migrating FANCI protein in the F010191 LCLs resulting from the missing residues from the C-terminus. (C) Equal amounts of fractionated cytoplasmic (C) and nuclear (N) protein extracts from normal or FA-I lymphoblast lines were analyzed by Western blot with the indicated antibodies. Procedure for fractionation is described in “Subcellular fractionation.” (D) Nuclear accumulation of FANCI and FANCD2 proteins was quantified by determining the band intensity of underexposed Western blot film with ImageJ Version 1.43 software. Error bars represent SD from 3 independent experiments. The percentage of protein in the nucleus is compared with total from both cytoplasmic and nuclear fractions.