Menin Is Phosphorylated in Response to DNA Damage Figure 7. Menin does not colocalize with c-H2AX foci. Immunofluorescence was performed in HeLa cells following treatment with 1000 Rads c-IR (6 hr), 25 J/m2 UV (2hr) or 2 mM HU (18hr) and colocalization of menin with c-H2AX (A) and SC35 (B) was assessed. doi:10.1371/journal.pone.0016119.g007 phosphorylation sites have remained elusive. Complete elucidation of this question is likely dependent upon a cellular system that faithfully recapitulates the DNA damage response and contains matched cells with or without menin. Materials and Methods Cell Culture and treatments 293T, HeLa, U2OS (ATCC), bTC3 [36], and Men12/2 MEF cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 1% Pen/Strep and 1% L-Glu. Lymphoblastoid (Coriell Cell Repository) cells were cultured in RPMI supple- mented with 20% FBS, 1% Pen/Strep and 1% L-Glu. Gamma Irradiation treatment was performed using Cs137 source. UV treatment was performed using a Stratalinker 2400 (Stratagene). Adriamycin (Fisher) was used at 0.05 uM for 18 hours and hydroxyurea (Sigma) was used at 2 mM for 18 hours. KU55933 and DNA-PK Inhibitor II (EMD Biosciences) were used at 10 uM and added 2 hours prior to treatment with 1000 Rads c-IR or 25 J/m2 UV. Cycloheximide (Sigma Aldrich) was used at 20 ug/ ml and timepoints were harvested in lysis buffer. Vectors and transient transfections pCDNA-Flag wildtype menin and MEN1 missense point mutants have been described [12]. pCDNA-Flag wildtype menin was used as a template to generate Ser-to-Ala mutants using Stratagene Site Directed mutagenesis. All mutations were confirmed by sequencing. 293T or HeLa cells were transfected PLoS ONE | www.plosone.org 10 January 2011 | Volume 6 | Issue 1 | e16119