Fig. 5

(A) Schematic diagram of FANCI highlighting the overlapping FANCD2- and PIDD1- binding domains (D2BD and P1BD, respectively; see Figure 2G?H).

(B) HeLa cells transfected with fixed or increasing amounts of expression vectors encoding HA-FANCI, Flag-PIDD1-FL and GFP-FANCD2, as indicated. Lysates were harvested 24 hrs post transfection and HA IPs were analyzed by western blot.

(C) HeLa cells stably expressing the indicated shRNAs or transfected with indicated siRNAs were synchronized as in Table S1B, treated with or without Gö6976 and MMC (1 ?M each) and harvested at 9 hrs post-treatment. FANCI IPs were analyzed by western blot

(D) HeLa cells synchronized as in Table S1A were treated with or without Gö6976 and MMC (1?M each), harvested at indicated time points post-treatment, stained with phospho-Histone H3 antibody and analyzed by flow cytometry.

(E) FANCI IPs from HeLa cells as in (D) were analyzed by western blot. hpt, hours post-treatment; int, interphase; M, mitosis.

(F) HeLa cells transfected with indicated siRNAs and treated with or without MMC (1 ?M) were harvested at 24 hrs. FANCI IPs were analyzed by western blot

(G) HeLa cells synchronized as in Table S1A were treated with or without RO-3306 (10 ?M) or nocodazole (200 ng/ml) 1 hr after release. Cells were then treated with Gö6976 (1 ?M) and MMC (1 ?M) and harvested 9 hrs later. FANCI IPs were analyzed by western blot.

(H) Schematic summarizing the results in panels D-G.