Fanconi anemia (FA) is a recessively inherited disease characterized in the cellular level by spontaneous chromosomal instability and specific hypersensitivity to cross-linking agents. pH 8/10% sucrose/1% Triton X-100/0.2% SDS/5 mM DTT/1 mg/ml protease inhibitor (Pefabloc, Boehringer Mannheim)] containing lysozyme (Sigma) and sonification, GST-FANCA1C271 (and were stained with … Co-IP of Transiently Expressed FANCA and FANCG Proteins. Because the localization of FANCG resembles that of FANCA (17, 18), epitope-tagged versions of FANCA and FANCG were used to explore potential interaction between these proteins. Vectors encoding functionally active FANCA-FLAG and HA-FANCG were transiently transfected into 293-EBNA Plinabulin cells. IP of FANCA-FLAG (and were studied to determine the effect of these mutations on the FANCA/FANCG Plinabulin interaction. Similar to HSC72, no FANCA was detected after precipitation of FANCG from cell line EUFA268 (Fig. ?(Fig.55= 3) reduced when coprecipitated with FANCG (Fig. ?(Fig.66and together with either full-length FANCA or a panel of Plinabulin carboxyl-terminal deletion constructs, in the presence of [35S]methionine. HA-FANCG PSTPIP1 was found to coprecipitate with both full-length FANCA and all of the carboxyl terminal-truncated FANCA proteins tested, the smallest of which comprised the amino-terminal 300 residues of FANCA (Fig. ?(Fig.77and labeled with [35S]methionine. (through XPG, all leading to an extreme sensitivity to sunlight (UV) and skin abnormalities. Proteins defective in the various complementation groups are all components of the nucleotide excision repair machinery (35). Even though Plinabulin the molecular role of the FA proteins remains to be established, the finding of a presumed functional complex and direct interaction between the FANCA and FANCG proteins may serve as an important foothold to further elucidate the FA pathway. Specifically, mapping of the exact binding domains in FANCA and FANCG may reveal structural clues about the nature of the interaction and its relationship to the function of other proteins participating in the FA pathway. In addition, the existence of a functional FA protein complex may have important implications for experimental systems that call for the introduction of a drug-sensitive cellular phenotype, such as cancer gene therapy. For example, overexpression of peptides that mimic either binding domain may block complex formation and cause a cell to become FA-like, i.e., apoptosis-prone and hypersensitive to cross-linking chemotherapeutic agents, such as mitomycin C, cyclophosphamide, and cisplatinum. Acknowledgments We thank A.B. Oostra, F.M. di Summa, C.G.M. van Berkel, M.A. Rooimans, and N. Cool for establishment and characterization of lymphoblastoid cell lines; Y. Waterham-de Vries for technical assistance; G.C. Ricotti for the topoisomerase II- mAb; C.G. Mathew for providing cell line BD32; M. Grompe for cell line PD20; and M. Buchwald for the HSC cell lines. This work has been supported by the Dutch Cancer Society (VU97C1565) and the Fanconi Anemia Research Fund. H.Y. and F.A.E.K. are supported by a grant from the National Institutes of Health (HL52138), and M.E.H. is supported by National Institutes of Health Grant HL56045. ABBREVIATIONS FAFanconi anemiaGFPgreen fluorescent proteinHAhemagglutininGSTglutathione S-transferaseIPimmunoprecipitation.