Mitotic phosphorylation of ATF2 and ATF7 by Cdk1.

<p>(<b>A</b>) Cells were synchronized at S, G2, or M phases. Mitotic cells were collected by mitotic shake-off, and whole cell lysates were analyzed by WB using antibodies against ATF2/ATF7 (ATF2[N96]), pATF2/pATF7 (pT69/71), cyclin B1, and actin. (<b>B–D</b> Cells were arrested at G2 phase using 9 µM RO-3306 and released into RO-3306-free medium containing 10 µM MG132. (<b>B</b>) At 20 min after release, cells were treated for an additional 60 min in the presence of 10 µM MG132 together with DMSO (solvent control) or 9 µM RO-3306. Cells were doubly stained with anti-pT69/71 antibody and PI. Cells stained with anti-histone pT69/71 antibody are pseudo-colored as green. Scale bars, 20 µm. (<b>C</b>) At 20 min after release from RO-3306, cells were treated with DMSO (solvent control), 9 µM RO-3306, 1 µM MLN8237, 10 µM ZM447439, 1 µM BI2536, 20 µM SB202190, 20 µM SP600125, or 20 µM U0126 for a further 30 min in the presence of 10 µM MG132. Whole cell lysates were analyzed by WB. Asyn, asynchronous. (<b>D</b>) At 20 min after release, cells were treated for an additional 30 or 60 min in the presence of 10 µM MG132 together with DMSO (solvent control), 9 µM RO-3306, 10 µM Gö6976, or RO-3306 plus Gö6976. Whole cell lysates were analyzed by WB. Asyn, asynchronous. Full-length blots are presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s007" target="_blank">S7B</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s008" target="_blank">S8A–B</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116048#pone.0116048.s009" target="_blank">S9 Figs</a>.