The interaction between FANCA and FANCL is dependent on other FA proteins and detected in the nucleus. (A) Whole-cell extracts of 10 million lymphoblastoid cells were immunoprecipitated with an antiserum against the N-terminus of FANCA (amino acid 1-271). After blotting, membranes were probed with α-FANCA antibody (Rb89) and α-FANCL antibody¹²  to show the interaction between FANCA and FANCL. A direct FANCL Western on whole-cell extracts (WCE) of 500 000 lymphoblastoid cells was performed to show the total cellular FANCL levels. (B) Whole-cell extracts of 10 million lymphoblastoid cells were immunoprecipitated with an antiserum against the C-terminus of FANCG (amino acid 480-622). After blotting, membranes were probed with α-FANCA antibody (Rb89), α-FANCB antibody,³  α-FANCG antibody (Rb43), and α-FANCL antibody¹²  to show the interaction between FANCA, FANCB, FANCG, and FANCL. (C) Whole-cell extracts of 10 million lymphoblastoid cells were immunoprecipitated with an antiserum against the N-terminus of FANCM (amino acid 1-70). After blotting, membranes were probed with α-FANCM antibody,¹³  α-FANCA antibody (Rb89), and α-FANCL antibody¹²  to show the interaction between FANCA, FANCL, and FANCM. (D) Mammalian 3-hybrid assay in 293 cells expressing FLAG-FANCB. Cells were cotransfected with the indicated protein pairs and assayed for luciferase activation. Fold induction relative to reporter gene activation when full-length FANCL is expressed alone. Error bars represent SD of 3 experiments. (E) Nuclear and cytoplasmic fractions of lymphoblasts cell lines were immunoprecipitated with an anti-FANCA antibody (as described for panel A). In addition, aliquots of protein lysate were probed with anti–beta tubulin and anti–TOPO I to check integrity of fractionation procedure.