conditions, incubated on ice for 30 minutes and spun at 4uC for 5 minutes at 14,000 rpm. Supernatant was transferred to a new tube and protein was quantitated with Bradford protein assay (BioRad, Hercules, CA, USA). For the breast tumor tissue lysis, the tissue was ground with a mortar and pestle in the presence of liquid nitrogen to create a powdered tissue. RIPA buffer (50 mM Tris- HCl pH 7.5, 105 mM NaCl, 1% NP-40, 1% sodium deoxycho- late, 0.1% SDS, 2 mM EDTA) was added to the powdered tissue, the sample vortexed for 60 seconds, and placed on ice for 45 minutes. Samples were homogenized with a syringe and needle, followed by centrifugation at 14,000 g for 10 minutes. Superna- tants containing the lysate were quantitated with Bradford protein assay (BioRad). Cell Synchronization MDA-MB-231 cells were synchronized by treatment with nocodazole (200 ng/ml) for 24 hours. Following treatment, arrested cells were collected by the mitotic shake method. Adherent cells were not collected. One fourth of the cells were collected (M phase population) and the remaining cells were released in Dulbecco’s modified Eagle’s medium supplemented with 20% FBS, 1% L- glutamine, and 1% streptomycin/penicillin. Cells were collected at 4 hours (G1 phase), 12 hours (S phase), and 20 hours (G2/M phase). Whole cell protein extracts from synchronized MDA-MB- 231 cells were separated on a 4–20% gel by SDS-PAGE, and western blotted with antibodies against Cyclin B(H-433), Cyclin D1(M-20), Cyclin E(C-19), cdk4(H-303) and actin(C-11). Western blot analysis Cell extracts were resolved by SDS PAGE on a 4–20% tris- glycine gel (Invitrogen, Carlsbad, CA, USA). Proteins were transferred to polyvinylidene difluoride microporous membranes using the iBlot dry blotting system as described by the manufacturer (Invitrogen). Membranes were blocked with Dul- becco’s phosphate-buffered saline (PBS) 0.1% Tween-20+3% BSA. Primary antibody against specified proteins was incubated with the membrane in blocking solution overnight at 4uC. Western blots were performed with a 1:1 of anti-dimethyl arginine (7E6) (Novus, Littleton, CO, USA) and anti-methyl mono/di arginine (AbCam, Cambridge, MA, USA), anti-methyl lysine (AbCam), anti-PRMT1 (Cell Signaling, Danvers, MA, USA) and anti- BRCA1(C-20), anti-Sp1(16C) and anti-STAT1 (E-23) (Santa Cruz, Santa Cruz, CA) antibodies. Membranes were washed twice with PBS+0.1% Tween-20 and incubated with HRP- conjugated secondary antibody for one hour in blocking solution. Presence of secondary antibody was detected by SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL, USA). Luminescence was visualized on a Kodak 1D image station. Immunoprecipitation assay For immunoprecipitation (IP) 1–4 mg of whole cell protein or tumor tissue extracts were brought up to a final volume of 500 ml with lysis buffer and precleared for 30 minutes with 50 ml of 30% A/ G agarose bead slurry (CalBioChem, La Jolla, CA). Supernatants were transferred to a new tube with 10 mg of BRCA1 or normal rabbit IgG antibodies (Santa Cruz), and the solution was rotated overnight at 4uC. The next day complexes were precipitated with A/G beads for 90 minutes. Beads were washed twice with TNE150+0.1% NP-40 and once with TNE50+0.1% NP-40. GST pull-down and in vitro methyltransferase assay GST tagged proteins were purified as described previously [53]. Constructs were washed three times with PBS+1% Triton X-100, BRCA1 Methylation pelleted and resuspended in the methyltransferase buffer reaction. Five hundred nanograms of GST-BRCA1 1–500, 452–1079, 504– 802, 697–1276, 1021–1552, 1501–1861 and core histones were incubated with 0.2 mg of recombinant purified PRMT1 (Active Motif, Carlsbad, CA, USA) in the presence of 0.55 mCi S- Adenosyl-L-[methyl-3H] methionine (GE Healthcare, Piscataway, NJ, USA) and reaction buffer (50 mM Tris-HCl pH 8.0, 20 mM KCl, 10 mM MgCl2, 250 mM sucrose, 10 mM b-mercaptoetha- nol) overnight at 37uC in a final reaction volume of 20 ml. The overnight methylation reactions (beads containing substrate) were spun, washed three times in excess cold 10% TCA, 1% sodium phosphate followed by once with 100% ethanol. Control samples were spotted on GF/C membranes (Millipore, Bedford, MA, USA), allowed to dry and processed equally as beads alone. Both beads and filters were counted in Beckman Coulter LS6001C scintillation counter in 2 ml of scintillation fluid (Beckman Coulter, Fullerton, CA, USA). For the protein GST-BRCA1 pull-down, 2 mg of HeLa whole cell protein extract were brought up to a final volume of 500 ml with lysis buffer and 500 ng of GST-BRCA1 constructs were rotated at 4uC overnight. Beads were washed twice with TNE150 + 0.1% NP-40 and once with TNE50 + 0.1% NP-40. siRNA-mediated knockdown of PRMT1 in HeLa cells HeLa cells (1.86107 cells) were transfected with double- stranded duplex with Hs_HRMT1L2_7 HP Validated siRNA (Qiagen, Valencia, CA, USA) or luciferase (Dharmacon, Lafay- ette, CO, USA) using Lipofectamine reagent according to the manufacturer’s recommendations (Invitrogen). Initial transfections were carried out in 24-well plates in order to establish optimal knockdown conditions by titrating siRNA at 10, 25 and 50 nM. Transfections for chromatin immunoprecipitation assays were conducted at 50 nM final siRNA concentration. Chromatin immunoprecipitation assay (ChIP) MDA-MB-231 cells were treated with 30 mM adenosine periodate methyltransferase inhibitor (AdOx, Sigma, St Louis, MO, USA) and processed 48 hours later for ChIP using an established protocol. Approximately 56106 cells were used per IP. Cells were cross-linked with 1% formaldehyde at 37uC for 10 minutes, pelleted, washed, and cells lysed using SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0, one tablet complete protease inhibitor cocktail per 50 ml) on ice for 10 minutes. Cells were sonicated on ice for 6 cycles to obtain an average DNA length of 500 to 1200 bp. Lysate was clarified by centrifugation at 4uC for 10 minutes at 14,000 rpm. Supernatant was then diluted 10 fold in ChIP dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris-HCl, pH 8.0, 167 mM NaCl) and pre-cleared with a mixture of protein A/G agarose (blocked previously with 1 mg/ml salmon sperm DNA and 1 mg/ml BSA, Stratagene, La Jolla, CA, USA) at 4uC for 1 hour. Pre-cleared chromatin was incubated with 10 mg of antibody at 4uC overnight. Next day, 60 ml of 30% slurry of blocked protein A/G agarose was added and complexes incubated for 2 hours. Immune complexes were recovered by centrifugation and washed once with low salt buffer (0.1% SDS, 1% Triton X- 100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 150 mM NaCl), twice with high salt buffer (0.1% SDS, 1% Triton X-100 2 mM EDTA, 20 mM Tris-HCl, pH 8.0, 500 mM NaCl), once with LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0), and once with TE buffer. Immune complexes were eluted twice with elution buffer (1% SDS, 0.1 M NaHCO3) and incubating at room temperature for 15 minutes on a rotating wheel. Cross-links were reversed by PLoS ONE | www.plosone.org 10 June 2010 | Volume 5 | Issue 6 | e11379