FANCA serine 1449 phosphorylation is dependent on ATR in vivo. (A) GM6914 cells expressing wild-type Flag-FANCA, Flag-FANCA (S1449A), or the vector control were treated with 0.1 μM MMC for 20 hours. At 16 hours, 1 μM wortmannin was added (lanes 2, 4, and 6) for 4 hours. Whole cell lysates were separated by SDS-PAGE and immunoblotted for phospho-FANCA S1449 and total FANCA. (B) Wild-type (GM02188), ATR-Seckel (DK0064), and ATM-deficient (GM01389D) cell lines were treated with 50 nM MMC and the indicated dose of wortmannin for 18 hours before preparation of lysates. Western blotting was performed with the phosphospecific or nonspecific antibodies to FANCA. P-S1449 indicates phosphoserine 1449.