Anti acetyl histone h3 lys9 lys14

The LNCaP cells were seeded in 100 mm dishes in phenol red-free RPMI containing 5% csFBS. On the next day, cells were treated with DHT or solvent for 6 hr. Chromatin immunoprecipitation (ChIP) assay was carried out using SimpleChIP Enzymatic Chromatin IP Kit (Cell Signaling Technology) according to manufacturer's protocol. Cells were fixed with 1% formaldehyde (Sigma Aldrich) for 15 min at 37°C and nuclei were prepared. Chromatins were digested with micrococcal nuclease to achieve a DNA ladder of 300-1,000 bp. The chromatins were immunoprecipitated with anti-AR (Abcam, Cambridge, MA, USA), anti-Histone H3, and anti-Acetyl-Histone H3 (Lys9/Lys14) (Cell Signaling Technology) antibodies overnight at 4°C. The precipitated DNA was used for PCR amplification with the following specific primer pairs to ARE regions: FKBP5 ARE (5'-GGAGCCTCTTTCTCAGTTTTG-3' and 5'-CAATCGGAGTGTAACCACATC-3'), DEPTOR promoter (5'-CCATAGAAAAAGATACAGGC-3' and 5'-TCATTCCATGAAATTTGAATG-3'), DEPTOR ARE1 (5'-TCCCCTCTTAATCTGGTTA-3' and 5'-CTAAT-GGCATACGGGCTTGT-3') and DEPTOR ARE2 (5'-TGAAGACGTTGAGGCATTTG-3' and 5'-ACAG-GGCCAAAGCATATGAG-3'). Band density was measured by ImageJ software.

Ejaculates were washed in 0.1 M PBS, pH 7.4, then dissolved in lysis buffer containing 7 mol/l urea, 2 mol/l thiourea, 4% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (Chaps), 65 mmol/l dithiothreitol (DTT), 1% (w/v) protease inhibitor cock-tail (Roche) for 45 min. The suspension was centrifuged at 16,000 g for 30 min at 4°C. The supernatant was quantified for protein content using bicinchoninic acid (BCA) method.
Equal amounts of protein were separated on 15% SDS-PAGE and transferred to a polyvinylidene fluoride membrane. Primary antibodies used were as follows:
anti-protamine 1 (1:1000; Santa Cruz Biotechnology), anti-protamine 2 (Santa Cruz Biotechnology), antiacetyl-histone H3 (Lys9/Lys14) (Cell Signaling) and polyclonal anti-acetyl-histone H4 (Millipore) antibodies (1:1000 dilution). The membranes were then incubated with the appropriate secondary antibody for 1 h at room temperature. The antigens were visualized by the electrochemiluminescence (Millipore, Darmstadt, Germany). The results were normalized by Gpadh levels.