using Fugene6 (Roche) using manufacturers protocol. Retroviral infections of Men12/2 MEFs were done as previously reported [12]. Antibodies Phospho-menin antibodies were generated by Bethyl laborato- ries and affinity purified. Total menin (A300-105A) antibody was also from Bethyl Laboratories. Flag M2 (F3165), Vinculin (V4505) and SC35 (S4045) were from Sigma. Phospho-Ser15-p53 (9284), and phospho-Ser139-H2Ax (2577) were from Cell Signaling. Total RNAPII-CTD (MMS-126R), pSer5-CTD (MMS-134R) and pSer2-CTD (MMS-129R) were from Covance. p53 clones DO1 (SC-126X) and FL (SC-55476) were from Santa Cruz Biotechnology. Histone H3 trimethyl K4 (ab8580) was from Abcam. Whole cell extracts and immunoprecipitations Extracts were prepared in IP lysis buffer as described in [12] containing Protease and Phosphatase Inhibitors (EMD Bioscienc- es) and lysates were sonicated 3 times at setting 1 for 5s pulses with a Branson Sonifier 450.Samples were centrifuged for 20 minutes at maximum speed and the supernatants moved to new tubes. Protein concentrations were measured and equal levels were used for each immunoprecipitation. Antibody and Protein G beads (Amersham) were added to each supernatant and rocked with constant motion for 3 hours and then washed 4 times in 1 ml of IP lysis buffer. The resulting immunocomplexes were resuspended in 50 ul of 1X SDS loading buffer, boiled for 5 minutes and run on 4-20% Tris-Glycine gels (Invitrogen). The gels were transferred to nitrocellulose and probed with indicated antibodies. Immunoprecipitation histone methyltransferase assay These studies were performed as described [12]. Immunofluorescence These studies were performed as described [37]. Real time quantitative PCR RNA was purified using RNeasy Columns (Qiagen) with DNase digestion. Superscript III RT (Invitrogen) and random hexamers were used to make cDNA. The Applied Biosystems 7500 real time PCR machine was used with Applied Biosystems Power Sybr master mix using an anneal/extend temperature of 60u. Relative expression was calculated using DDCt. Statistical significance was calculated by two-tailed T-test comparing DNA damage with untreated conditions. Primers used for qRT-PCR are listed in Table S1. Chromatin Immunoprecipitation ChIP was performed and analyzed as described [38], with the following modifications, chromatin was sheared using a Branson Sonifier 450 at power setting 4, 30% duty cycle for 861 min pulses to generate 200–600 base pair fragments, crosslinking was reversed overnight at 65uC in elution buffer, supernatant was diluted in an equal volume TE buffer and digested with Proteinase K for 2 hr at 37uC. The DNA was purified using PCR Cleanup Columns (Qiagen) following manufacturers protocol and diluted 4 fold in TE. Quantitative PCR was performed in triplicate. A 1:1 mixture of p53 DO1 and FL antibodies were used for p53 IPs. Primers for the CDKN1A and MDM2 [39], GADD45A [34]and also are listed in Table S1. The Applied Biosystems 7500 real time PCR machine was used with Applied Biosystems Power Sybr master mix using an anneal/extend temperature of 60u All PCR Menin Is Phosphorylated in Response to DNA Damage reactions were run in triplicate and the average Ct value was to calculate the percent input recovered using the formula = 2^(average input Ct-average antibody Ct). Statistical significance was calculated by a two-tailed T-test comparing the percentage of input DNA recovered in each immunoprecipitation with the no antibody control. All PCR primers amplified a single product of the appropriate size. Supporting Information Figure S1 Trimethylation of histone H3 is not signifi- cantly increased after IR. ChIPs were performed in U2OS cells 6 hours following treatment with 1000 Rads of c-IR and immunoprecipitated with an antibody recognizing histone H3K4me3. Precipitated chromatin was used for quantitative real-time PCR using primers that amplify 59 regions of the indicated genes. Results are the average of at least 3 independent experiments and represent the amount of DNA immunoprecip- itated with each antibody relative to the amount of input DNA. Error bars indicate standard error of the mean. (TIF) Figure S2 Menin mass spectrometry data. (A) Endoge- nous menin was immunoprecipitated from untreated 293T cells or 18 hours after addition of 2 mM Hydroxyurea, 6 hours after 1000 Rads of c-IR, or 2 hours after exposure to 25 J/m2 UV. The identified phospho-peptides are listed by treatment conditions with the predicted Serine site in bold. Coverage indicates peptides corresponding to total protein analyzed by mass spec. (B) Endogenous menin was immunoprecipitated from the mouse b cell line bTC3. The identified phospho-peptides are listed. (C) Alignment of identified phosphorylation sites with menin sequenc- es from other metazoans using NCBI COBALT alignment software. (TIF) Figure S3 Menin phosphorylation after DNA damage. (A) Flag IPs from 293T cells transfected with Flag-wildtype menin, or Flag-phospho-deficient mutants harvested 2 hours after treat- ment with 1000 Rads of c-IR. IPs were resolved and immuno- blotted with phospho-Ser394, phospho-Ser543 or total menin. (B) Endogenous menin was immunoprecipitated from untreated 293T cells or 2 hours after treatment with 25 J/m2 UV, 18 hours after addition of 2 mM Hydroxyurea, 18 hours after addition of 0.05 uM Adr, or 2 hours after 1000 Rads of c-IR. Immunopre- cipitates were resolved and immunoblotted with phospho-specific antibodies. (B) Time course immunoprecipitations of Flag-menin wildtype or Flag-Ser487Ala mutant after 1000 Rads of c-IR or 25 J/m2 UV treatment and immunoblotted with phospho-specific antibodies. (C) Time course immunoprecipitations of Flag-menin wildtype or Flag-Ser394Ala mutant after 1000 Rads of c-IR or 25 J/m2 UV treatment and immunoblotted with phospho-specific antibodies. (D) 293T whole cell extracts from cells treated with 1000 Rads of c-IR in the presence or absence of 20 ug/mL CHX and immunoblotted for menin and Vinculin as a loading control. (TIF) Figure S4 Menin Ser-to-Ala mutant phosphorylation kinetics. (A) Time course immunoprecipitations of Flag-menin wildtype or Flag-Ser487Ala mutant after 1000 Rads of c-IR or 25 J/m2 UV treatment and immunoblotted with phospho-specific antibodies. (B) Time course immunoprecipitations of Flag-menin wildtype or Flag-Ser394Ala mutant after 1000 Rads of c-IR or 25 J/m2 UV treatment and immunoblotted with phospho-specific antibodies. (C) 293T whole cell extracts from cells treated with PLoS ONE | www.plosone.org 11 January 2011 | Volume 6 | Issue 1 | e16119