S-phase–specific monoubiquitination of the FANCD2 protein.

(A) HeLa cells were synchronized by the double-thymidine–block method. Cells corresponding to the indicated phase of the cell cycle, as determined by flow cytometric analysis of DNA content, were lysed and processed for FANCD2 immunoblotting. FANCD2-L is the monoubiquitinated isoform, and FANCD2-S is an unubiquitinated isoform. Immunoblots of FANCD2 during cell-cycle progression were also analyzed following synchronization by either nocodazole block (B) or mimosine block (C). (D) HeLa cells were blocked in M phase using nocodazole and subsequently released into drug-free medium to allow cell-cycle progression. At the indicated times following release (corresponding to the indicated phase of the cell cycle), cells were fixed, immunostained with anti-FANCD2 antibody, and analyzed by immunofluorescence microscopy. (E) Analysis of FANCD2 immunolocalization in HeLa cells synchronized with mimosine. Original magnification, × 600.