Figure 6. Phosphorylation of S347 may be required for proper activation of the FA/BRCA pathway upon DNA damage.
A. Monoubiquitination of FANCD2 is inhibited in U2OS cells stably expressing the FANCA S347A mutant. U2OS cells stably expressing HA-FANCA WT (WT#5) and the S347A mutant (S347A#6 and S347A#20) or empty vector-transfected cells (pcDNA3#2) were treated with 25 ng/mL MMC for 8 h. Monoubiquitination of FANCD2 was visualized as described in Figure 2A. B. Formation of FANCD2 nuclear foci is inhibited in cells that stably overexpress FANCA S347A. FANCD2 nuclear foci were visualized as described in Figure 2B. Representative images of MMC-treated samples are shown in the left panel. The number of foci per cell was counted and plotted (right panel). The values represent the mean ± SEM. (Student's t-test, **, P < 0.01).