FANCA transduction enhances FA protein complex stability and restores nuclear FANCA levels. (A) Whole cell lysates. All 4 rows depict data obtained from the same filter, cut horizontally at 80 kDa to allow FANCA to be interrogated on the top portion and FANCC (then FANCG after stripping the FANCC signal) on the bottom portion. Retroviral transduction with each of 3 FA cDNA constructs resulted in increased levels of their respective proteins (the results from independently obtained duplicates are shown for each transduced cell line). Transduction with FANCA (UoC-M1/FA) also led to an increase in cellular levels of FANCG, whereas the reverse was not observed (ie, transduction with FANCG [UoC-M1/FG] did not increase cellular levels of FANCA). Row 4 is a constant, nonspecific fragment detected by the FANCG antibody and demonstrates equal loading in all lanes. (B) Nuclear extracts from 3 transduced lines (UoC-M1/FA, UoC-M1/FC, and UoC-M1/FG), and the myeloid cell line Mo7e probed with anti-FANCA (row 1) and anti-FANCG (row 2) antisera. Transduction with FANCA restored nuclear levels of FANCA in UoC-M1/FA (row 1, column 2), whereas transduction with FANCG did not increase nuclear levels of FANCG (UoC-M1/FG) (row 2, column 5). (C) Nuclear levels of FANCA and FANCG are not reduced in the lymphoblastoid cell line 4081. The cell line JY is an EBV-transformed lymphoblastoid cell line established from a healthy adult volunteer and also demonstrates cellular resistance to MMC. Lane 1 is a whole cell extract (WCL) from HeLa cells.