Monoubiquitination is required for targeting of FANCD2 to the chromatin fraction. PD-20Fs (FA-D2 fibroblasts) were stably transduced with retroviral constructs encoding HA-FANCD2-44 (wild type), HA–Exon44-T, HA–mutant EDGE, or HA–FANCD2-K561R. Cells were pretreated with MMC to increase the level of FANCD2-L to approximately 50% of total cellular FANCD2. Cells were then fractionated as described in Figure 5A, and fractions were immunoblotted with antisera to FANCD2, histone H4, or lamin B. Fractions are shown beginning with the detergent-insoluble pellet following permeabilization (P2).