FANCD2 carboxy terminal mutants are monoubiquitinated following ionizing radiation. (A) FA-D2 fibroblasts were stably transduced with either hemagglutinin (HA)–pMMP (empty vector), HA-FANCD2 (wild type), HA–Exon44-T, or HA–mutant-EDGE, as indicated. Cells were irradiated with 15 Gy, as indicated, and whole-cell lysates were immunoblotted with anti-FANCD2 antisera. (B) Immunofluorescence of the transduced cells 8 hours after cellular exposure to IR, using an anti-FANCD2 antiserum. (C) Colocalization of FANCD2 and phosphorylated histone H2AX in DNA damaged-inducible foci. HeLa cells were either untreated (top) or exposed to IR (15 Gy, 15 hours) (bottom), as indicated. HeLa cells were double-stained with monoclonal anti-FANCD2 (FI-17; green) and polyclonal anti–histone γ-H2AX (Ser139; red). Merges of corresponding anti-FANCD2 and anti–histone γ-H2AX images are shown. Original magnifications × 630 (B-C).