Monoubiquitinated FANCD2 is preferentially retained in the chromatin fraction. (A) Protocol for nuclear fractionation of transduced FA-D2 fibroblasts. Cytoplasm and nucleoplasm were extracted by permeabilization with detergent, and resulting nuclei were nuclease-digested and extracted with NH₂SO₄. Supernatants (S) and pellets (P) were analyzed for FANCD2, histone H4 (chromatin marker), and lamin B (nuclear matrix marker) by Western blot. (B) Monoubiquitinated FANCD2 is preferentially retained in detergent-insoluble nuclei (P2) and is subsequently extracted with chromatin (S4). (C) FANCD2 nuclear foci are extracted with the chromatin fraction. HeLa cells were grown on coverslips and exposed to IR (15 Gy). After 12 hours, cells were prepared for immunofluorescence microscopy with an antibody to either FANCD2 or lamin B, as indicated. Immunofluorescence microscopy was performed on either unpermeabilized (whole) cells (equivalent to P1 fraction), cells permeabilized with Triton X-100 (equivalent to P2), or cells permeabilized and extracted with DNase I and ammonium sulfate (equivalent to P4 fraction). FANCD2 and lamin B images are from different representative cells. Original magnification × 630.