FANCA is induced after MMC treatment but is not phosphorylated during S phase. (A) Exponentially growing HeLa cells were treated with 1 μM MMC for the time indicated, before preparation of whole cell lysates and SDS-PAGE electrophoresis. Western blotting was performed with the phospho-specific or nonspecific antibodies to FANCA as indicated. (B) Conditions were as in panel A, with MMC treatment continued for the indicated time. (C). HeLa cells were synchronized by double thymidine block at the G₁/S border (lane 4) and released for 3 hours into S phase (lane 5) or 6 hours into late S/G₂ phase (lane 6). Alternatively, cells in lane 2 were treated with 1 μM MMC for 18 hours. Whole cell extracts were separated by SDS-PAGE and immunoblotted for phosphoserine 1449, FANCA, FANCD2, and Ku86 as a loading control. P-FANCA indicates phospho-FANCA.