Functional analysis of missense and other nontruncating FANCA mutations leading to an altered FANCA protein. (A) Analysis of FANCA expression by Western blot of patient-derived LCLs. Two cell lines with 2 truncating mutations (FA55 and FA178) and a cell line with a large deletion and a splicing mutation (FA145) are included as negative controls. A total of 40 μg of total extract was loaded per lane, except for lane 2, where 2 μg of wild-type (WT) cell extract was loaded to detect a minimum amount of FANCA protein. (B) Quantification of FANCA expression by densitometry, relative to the WT. (C) Analysis of FANCD2 monoubiquitinylation in patients' LCLs treated with 2mM HU for 24 hours. (D) FANCA subcellular localization analysis by sample fractionation and Western blot after treatment with 2mM HU for 24 hours. NF indicates nuclear fraction; and CF, cytoplasmic fraction. Cell-equivalent volumes from each cell fraction were loaded. ORC2 detection is included as a nuclear protein control and GAPDH as a cytoplasmic control. (E) Analysis of FANCA subcellular localization analysis and foci formation by immunohistochemistry after HU treatment. A wild-type and a representative cell line with missense mutations (FA170) are shown. Images were observed with an Axio Observer A1 microscope (Zeiss) using a 100×/1.3 oil objective. The slides were mounted with Vectashield (Vector Laboratories) and were captured with an AxioCam MRm camera (Zeiss). Digital images were aquired with Axiovision 4.6 software.