The CUE domain is required for FANCD2 and FANCI nuclear foci formation and chromatin localization. (A) FA-D2 cells reconstituted with WT or mutant FANCD2 were untreated or treated with 250nM MMC for 12 hours and then fixed and immunostained with antibodies against FANCD2 and FANCI. Nuclear foci were analyzed using a Zeiss AxioImager.A1 upright epifluorescent microscope with AxioVision LE 4.6 image acquisition software. The percentage of nuclei with greater than 5 foci were scored and plotted in the indicated histograms (***P < .001). (B) FA-D2 cells reconstituted with WT or mutant FANCD2 were untreated or treated with 250nM MMC for 12 hours, released from treatment and pellets collected at the indicated time points. Pellets were divided and one-half was lysed in 2% SDS lysis buffer, sonicated, and boiled (WCE). The remaining pellet was sequentially lysed in cytoskeletal buffer to extract soluble proteins (C) and then 2% SDS lysis buffer with sonication and boiling to extract proteins bound to chromatin (N). Proteins were resolved and immunoblotted with antibodies against FANCD2, FANCI, α-tubulin, and H2A.