Anti h3k9me1

ChIP assay was performed as we previously described [12 (link)]. Briefly, liver tissues were minced and crosslinked with 1% formaldehyde, then quenched with 125 mM glycine. After washed with cold PBS, samples were resuspended with digestion buffer plus 1 mM PMSF. Crosslinked chromatin was sheared with Micrococcal Nuclease (New England Biolabs, Beverly, MA) and sonication. Chromatin was immunoprecipitated using anti-H3K9me1, anti-H3K9me2, and anti-G9a (all from Abcam, Cambridge, MA) or respective IgGs (Sigma, MO; or Santa Cruz, CA). The immune-complexes were captured with Protein G agarose beads (GE Healthcare, Chicago, IL). ChIP-enriched and input DNA were extracted and analyzed by qPCR, with the inputs as the internal control. Primers for ChIP assay were provided in Table S1. Regions of GSTP1/2 promoter used for ChIP assay were ranged from −2000 bp to TSS (transcription start site), which was downloaded from http://genome.ucsc.edu/cgi-bin/hgNear. This region was averagely divided into four parts to cover the promoter area by one set of primer per 500 bp, and the primers (Table S1) were randomly but not selectively designed by Primer 5.

For the methyl-lysine pulldown assay, GST-3xMBT protein (the GST-3xMBT plasmid is a gift from Dr. Or Gozani) was expressed in E. coli DH5α cells and purified by glutathione Sepharose 4B chromatography following the manufacturer's protocol (Amersham Biosciences). Nuclear extracts from VCaP cells treated with or without 1 mmol/L pargyline were used for the pulldown assay with GST-3xMBT protein and the immunopurified proteins were separated by SDS-PAGE gel, stained with Coomassie blue, and then subjected to mass spectrometry analysis. For other immunoprecipitation (IP) assays, cells were lysed in Triton Lysis buffer and treated with protein inhibitor cocktails (Thermo Fisher Scientific), followed by a brief sonication, and then lysates were immunoprecipitated with anti-V5-conjugated beads (Sigma, A7345) or anti-FLAG-conjugated beads (Sigma, A2220). For immunoblotting, proteins were detected with primary antibodies, including anti-V5 (Abcam, ab9116), anti-EHMT1 (Abcam, ab41969), anti-EHMT2 (Novus, NBP2-13948), anti-FLAG (Sigma, F3040), anti-LSD1 (Abcam, ab17721), anti-CoREST (Abcam, ab183711), anti-HDAC1 (Abcam, ab7028), anti-HDAC2 (Abcam, ab7029), anti-REST (Millipore, CS200555), anti-E2F1 (Cell Signaling Technology, 3742), anti-GAPDH (Abcam, ab8245), anti-H3K9me1(Abcam, 9045), anti-H3K9me2 (Cell Signaling, 9753S), and anti-Tubulin (Abcam, ab6046).