Phospho-FANCA serine 1449 is induced after DNA damage. (A) HeLa cells treated with 1 μM MMC for 18 hours (lanes 2-5) and 1 μM okadaic acid for 30 minutes (lane 3) were lysed, and 175 μg protein was subjected to SDS-PAGE and immunoblot for endogenous FANCA. In lanes 4 and 5, 275 μg protein from HeLa cells treated with MMC was treated directly with λ-phosphatase and run on SDS-PAGE. Proteins were immunoblotted using antiphosphoserine 1449 antibody. FANCA immunoblot shows even FANCA expression. β-tubulin shows equal loading. All lanes depicted are from the same gel, same exposure. (B) HeLa cells expressing Flag-tagged FANCA, Flag-FANCA (S1449A), or vector control were treated with MMC 1 μM for 18 hours, lysed, run on SDS-PAGE, and immunoblotted using the same antibodies as in panel A. (C) Cell lysates from cells in panel B were immunoprecipitated with anti-Flag antibody and subjected to SDS-PAGE and immunoblot using antiphospho-(Ser/Thr) ATM/ATR substrate antibody. IP indicates immunoprecipitation; Ppase, phosphatase; P-S1449, phosphoserine 1449; P-L(S/T)Q, phospho-leucine (serine/threonine) glutamine (ATM/ATR substrate); and MW, molecular weight marker.