Exogenous expression of FANCI R1299X mutant localizes to cytoplasm in U2OS cells. (A) Table showing FANCI and c-Myc NLS amino acid sequence used in the study. NLS software (www.predictprotein.org) was used to identify the putative C-terminal NLS sequence for FANCI. (B) Schematic diagram of FANCI WT, FANCI R1299X, and FANCI R1299X proteins with the fusion of NLS sequences from FANCI (FANCI-NLS2) or c-Myc (FANCI-NLS3) at the C-terminus and C-terminal NLS deleted (FANCI-ΔNLS). (C) U2OS were transfected with the indicated Flag-tagged FANCI constructs. Forty-eight hours after transfection, cells were fixed and stained with anti-Flag antibody. 4,6-diamidino-2-phenylindole is used for DNA staining in the nucleus. A total of 200 cells were counted and scored for predominantly nuclear (N), both nuclear and cytoplasmic (N-C), or cytoplasmic (C) staining and graphed as percentage of total cells. Representative images of each expression constructs are shown.