ntigen complexes from the first Ip were eluted by incubation in 10 mM DTT for 30 min at 37uC. Samples were diluted in 20 volumes 1% triton X-100, 2 mMEDTA, 150 mM NaCl, 20 mM Tris 8.0 and antibody added. Wash buffers were High salt, Low Salt, LiCl wash. Elution from beads was in 50 mM NaCl, 10 mM Tris 7.5, 5 mMEDTA with 0.5% or 1.2% SDS for 15minutes at room temp. DNA was purified with MinElute columns and quantified by Tideglusib cost fluorimetry with PicoGreen on a Synergy HT plate reader or by NanoDrop spectrophotometer. Non-immune rabbit IgG was used to determine nonspecific antibody interactions and was subtracted from specific interactions. Primers to an unrelated genomic region, ribosomal protein L30 or to 5S ribosomal DNA were used to determine that the changes seen were specific to the promoter. PCR Protocols ChIP DNA was analyzed by quantitative real-time with a TaqMan MGB Probe with a 6-FAM reporter and primers to MMTV Nuc B on a MJ Research Chromo4 RealTime PCR Detector DNA Engine. qPCR values were quantified by Absolute Standard Curve Method using plasmid pM25 , corrected for Input DNA and expressed as a ratio relative to untreated cells. Other mRNAs were quantified using SyberGreen and normalized to b-Actin mRNA by the Comparative C method after cDNA was made, referred to as reverse transcibed quantitative PCR. cDNA for PCR 22441874 was synthesized with MuLV reverse transcriptase, and Random hexamers. See Antibodies See Materials and Methods Cell Culture The 1470.2 cell line derived from the mouse adenocarcinoma parent line, C127i, constitutively expresses GR and has multiple copies of stably integrated MMTV-chloramphenicol acetyl transferase . Cells were grown in DMEM with either 10% calf serum, or charcoal stripped 1% CS for 1624 h prior to treatment with Dex 6sodium arsenite . Nuclear Extracts Mini-Dignam NEs were made. Cells were lysed in Buffer A, nuclei were isolated/extracted in Buffer C and dialysis was for 2 hr against Buffer D. Protein was measured by Bradford assay. Chromatin Immunoprecipitation Assays Cells were cross-linked with 1.5 mM Ethylene Glycol-bis at 25uC for 25 min followed by 1.0% formaldehyde at 25uC for 10 min or with formaldehyde only. Nuclei were isolated, and DNA digested to predominantly monosomes with micrococcal nuclease 37uC 13679187 for 6 min. ChIP Electromobility shift assays EMSAs included 20 mg of NE in Buffer D with 3060 fmol of -end-labeled wtGRE, 59-GATCCGGTacaATCtgtTCTA-39, and 0.2 mg poly.poly in 20 ml reactions separated on 5% acrylamide/0.5X Tris/Borate/ EDTA gels at 4uC. Imaging was by PhosphorImager analysis and quantification by ImageQuant software. Arsenic Inhibits CARM1 Gel Electrophoresis and Western Blot Analysis Proteins were separated on 10% polyacrylamide gels Pierce or NuPAGE Novex BisTris 420% gradient gels in recommended buffers, transferred to Immobilon-P PVDF membrane and visualized by HRP-based chemiluminescence imaged on film 
or on an Alpha Innotek FluoroChem 8900. Nuclear Run-ons Isolated nuclei were resuspended in 100 ml. One hundred microliters of 2x reaction mix was added with 120 mCi/200 ml reaction -UTP . and incubated for 45 min 30uC. RNA was extracted with TRI Reagent LS and equal counts were added to nitrocellulose filters previously blotted with PCR-amplified CAT reporter DNA, an equal amount of pUC18 DNA for background subtraction, and 5 s DNA for normalization. Hybridization was at 42uC in 50% formamide, 5x SSPE, 3x Denhardt’s, 100 mg/ml yeast tRNA and 0.5% SDS for 1216 h. Visualizatio
