Menin Is Phosphorylated in Response to DNA Damage the amount of DNA immunoprecipitated with each antibody relative to the amount of input DNA. Results are the average of at least 3 independent experiments. Error bars are standard error of the mean. * p,0.05. doi:10.1371/journal.pone.0016119.g003 phosphorylation sites were evaluated (Figure 4B). The Flag-tagged wild type, Ser487Ala and Ser543Ala menin expressing cells all showed a strong induction of Ser394 phosphorylation after DNA damage, with c-IR causing the strongest induction despite lower levels of immunoprecipitated menin as evaluated by total menin immunoblots. A low level of Ser394 phosphorylation was found in untreated cells. Phosphorylation of Ser487 was comparable between Flag-tagged wild type, Ser394Ala and Ser543Ala menin expressing cells. Investigation into the phosphorylation status of endogenous menin in 293T cells exposed to different forms of DNA damage confirmed the induction of Ser394 phosphorylation while the levels of Ser487 and Ser543 phosphorylation appeared to be constant across treatment conditions (Figure S3B). We next examined the phosphorylation kinetics of these sites following treatment with either c-IR or UV. Ser394 phosphorylation was detectable after only 15 minutes and remained elevated for at least 3 hours following c-IR (Figure 4C). UV treatment induced Ser394 phosphorylation first became detectable after 45 minutes and remained elevated for at least 3 hours. Ser487 phosphorylation was found to be more dynamic, with c-IR triggering a sharp increase followed by a gradual decline over time. UV treatment however, showed a peak of Ser487 phosphorylation by 30 minutes, followed by a rapid decrease in detectable levels. For comparison, Figure 4. Menin is phosphorylated following DNA damage. (A) Schematic diagram of menin showing locations of detected phosphorylation sites relative to nuclear export (NES) and nuclear localization sequences (NLS). (B) Flag IPs from 293T cells transfected with Flag-wildtype menin, or Flag-phospho-deficient mutants following treatment with 0.05 uM Adr, 25J/m2 UV or 1000Rads of c-IR. IPs were resolved and immunoblotted with phospho-Ser394, phospho-Ser487 or total menin. (C) Menin IPs were performed over the course of 3 hours from 293T cells treated with 1000 Rads of c-IR (left panel) or 25J/m2 UV (right panel) and immunoblotted with phospho-Ser394, phospho-Ser487 or total menin. Whole 293T cell lysates were probed for phospho-Ser15-p53 over the same time course (lower panel). doi:10.1371/journal.pone.0016119.g004 PLoS ONE | www.plosone.org 6 January 2011 | Volume 6 | Issue 1 | e16119