FIG 1.

FANCD2 is phosphorylated under nonstressed conditions and during the S phase of the cell cycle. (A) HeLa, U2OS, and COS-7 cells were incubated with or without 200 nM MMC for 24 h. Cells were harvested and lysed in lambda phosphatase lysis buffer and incubated in the absence or presence of lambda phosphatase and proteins analyzed by immunoblotting. (B) HeLa cells were synchronized at the G₁/S boundary by a double-thymidine block and released into thymidine-free media. Cells were lysed in lambda phosphatase buffer and incubated in the absence or presence of lambda phosphatase and lysates analyzed by immunoblotting. For cell cycle stage analysis, cells were fixed, stained with propidium iodide, and analyzed by flow cytometry. (C) FA-A (FANCA^−/−) and FANCA-complemented FA-A cells were synchronized at the G₁/S boundary by a double-thymidine block and released into thymidine-free media. Cells were harvested, lysed in lambda phosphatase buffer, incubated in the presence or absence of lambda phosphatase, and analyzed by immunoblotting.