Primary splenic lymphocytes from FA-G mice are defective in IR-inducible Fancd2 monoubiquitination.

(A) Schematic representation of the human FA pathway. The FA protein complex, containing the FANCA, FANCC, FANCF, FANCG, and perhaps FANCE proteins, is required for the monoubiquitination of the FANCD2-S protein (155 kd) to the FANCD2-L protein (162 kd). Monoubiquitination is increased in response to DNA damage by IR. Ubiquitinated (Ub) FANCD2 is targeted to foci containing the BRCA1 protein and other proteins involved in DNA repair. (B) Primary splenic lymphocytes (splenocytes) were isolated from mice with the indicatedFancg genotype. Cells were untreated or exposed to IR (2, 4, 10, or 20 Gy), as indicated. Cell lysates were prepared, and total cellular proteins were electrophoresed, transferred to nitrocellulose, and immunoblotted with an anti-Fancd2 antiserum. This polyclonal antiserum cross-reacts with the human and murine Fancd2protein and also recognizes a nonspecific 150 kd protein in both human and murine lysates, as indicated. Alternatively, whole tissue extracts were prepared from testes (lanes 17,18) from aFancg^+/− mouse or aFancg^−/− mouse. Cells examined were PD7 (wild-type human lymphoblasts), PD20 (human FA-D2 lymphoblasts), Ba/F3 (murine interleukin-3–dependent lymphocytes), or murine splenocytes (lanes 7-16). Both FANCD2-S and FANCD2-L isoforms are absent from the PD-20 (FA-D2) lymphoblasts, as previously described.