FANCM undergoes cell cycle–dependent phosphorylation. (A) Asynchronously growing cells (lane 1), cells arrested in G₁/S phase with double thymidine blocking and released into S phase (lanes 2-7), or cells arrested in metaphase with nocodazole and released into G₁ phase (lanes 8-12) were fractionated into S and P and immunoblotted for the indicated proteins. Asyn indicates asynchronous cells. Synchrony was monitored by flow cytometric analysis. The SDS-PAGE gel used here was 4% Tris-glycine. (B) Experimental protocol used to prepare cell extracts described in panels C and D. (C) Either exponentially growing Flag-FANCL HeLa cells (Asyn; lanes 1-4), cells released from double thymidine block for 3 hours (S phase; lanes 5-8), or cells arrested in mitotic phase with nocodazole (M phase; lanes 9-12) were fractionated into S100 and S300. As a negative control for immunoprecipitation with FLAG antibody, parental HeLa cells (lanes 13-16) were used. Each fraction was immunoprecipitated with FLAG-M2 agarose beads, and the precipitates were immunoblotted for the indicated proteins. (D) Cells were arrested in the G₁/S phase by double thymidine block (lanes 1-4) and released into S phase (lanes 5-16). Alternatively, cells were arrested in mitotic phase with nocodazole (laness 17-20) and fractionated into S100 and S300. Proteins from each fraction were immunoprecipitated with FLAG-M2 agarose beads, and the precipitates were immunoblotted with the indicated antibodies. Note that for the Western blots of Flag-FANCL (fourth row), immunoprecipitation samples were exposed for a shorter time than for input samples. This is indicated by the vertical lines in between. This was necessitated by the fact that the intensity of IgG bands that appeared on immunoprecipitation samples was substantially greater than that of the input Flag-FANCL bands. Entire images come from the identical gel.