Anti dimethyl histone h3 lys9

To evaluate transcription factor binding to key gonadotrope gene promoters, we performed ChIP assays. ChIP assays were performed as previously described [38 (link)]. Chromatin was sonicated to an average length of 300–500 bp using a Branson Sonifier 250 (Branson Ultrasonics Corp., Danbury, CT). Antibodies recognizing specific histone modifications were: anti-acetyl-Histone-H3 (06–599; Millipore, Temecula, CA), anti-trimethyl-Histone H3-Lys4 (07–473; Millipore), anti-dimethyl-Histone H3-Lys9 (ab1220; Abcam), all of which are ChIP-grade antibodies. To recognize phosphorylated polymerase, ChIP-grade anti-RNA polymerase II CTD repeat YSPTSPS (phospho S5) (ab5131; Abcam, Cambridge, MA), was used. Immunoprecipitated DNA and DNA from input chromatin were analyzed for sequences of interest by qRT-PCR using promoter-spanning primers specified in Table 1. For ChIP assays comparing αT1–1, αT3–1, and LβT2 chromatin, the percentage of enrichment of antibody signal over IgG was calculated for each primer set. IgG was the same species and source as the comparison antibody. Then, values for activating chromatin marks were normalized to those for the inactive gene Gnrh1. For repressive chromatin marks, values were normalized to the highly active gene Actb [37 (link)–39 (link)].

1 μg of isolated proteins underwent separation on a 15% or 10% SDS-PAGE prior to transfer onto PVDF membranes (BIORAD, Shanghai, China 162-0177), which then underwent a 2h blocking in 5% skimmed milk, followed by an ON incubation at 4 °C with anti-Histone H3 (Proteintech, 17168-1-AP, Planegg-Martinsried, Germany) (1:10,000), anti-Phospho-Histone H3 (Ser10) (Cell Signaling Technology, 13576S, Damvers, MA, USA) (1:1000), Anti-Di-Methyl-Histone H3 (Lys9) (Abcam, ab1220, Cambridge, UK) (1:2000), and anti-acetyl-Histone H3 (Lys18) (PTM BIO, PTM-158) (1:2000). This was followed by four PBST-rinses, and subsequent 2 h secondary antibodies incubation (Goat Anti-Rabbit IgG H&L (HRP, Abcam, ab6721, 1:10,000, Cambridge, UK) and Goat Anti-Mouse IgG H&L (HRP, Abcam, ab6789, 1:10,000), Cambridge, UK) at RT. Again, the membranes were rinsed four times in PBST buffer before employing the High-sig ECL Western Blotting Substrate (Tanon) to identify protein bands.