FANCL coimmunoprecipitates with FLAG-tagged FA proteins and is required for the assembly of the FA core complex. Lymphoblastoid cell lines stably expressing functionally active FLAG-tagged FANCA (A), FANCF (B), and FANCC (C) were generated. Whole-cell extracts of 10 million cells were immunoprecipitated with an α-FLAG affinity gel and precipitated proteins eluted by competition with a FLAG peptide. Untransfected cells (lane 1) were included as negative controls. Precipitated proteins were detected with α-FANCA (Rb89), α-FANCL,¹²  α-FANCF (Rb7), and α-FANCC (Rb39). In panel B, the bottom part of the blot was first probed for FANCL and then subsequently probed for FANCF, thus allowing detection of both proteins that run at a very similar molecular weight, 45 and 42 kDa, respectively. The asterisk in panels B and C mark a nonspecific polypeptide that cross-reacts with the FANCL antiserum. (D) Whole-cell extracts of 10 million WT, FA-L, and respective patient control lymphoblasts were immunoprecipitated using guinea pig antibodies against FANCC_6-105, FANCF_1-245, and FANCG_480-622. Precipitated proteins were detected using rabbit antibodies against FANCA (Rb89), FANCG (Rb43), and FANCF (Rb7). The FANCG protein is indicated with an asterisk. (E) Direct FANCC Western blot on whole-cell extracts of 500 000 lymphoblastoid cells to show the total cellular FANCC levels.