Anti ar

ChIP assays were performed according to previously described protocols (25 (link)). 22Rv1 cells were transfected with FLAG-BAP18 using jetPRIME transfection reagent (Polyplus) or infected with shBAP18, and then cultured for 2 days in phenol red-free RPMI 1640 supplemented with 10% charcoal-dextran-stripped FBS. At ∼90% confluency, cells were treated with 10⁻⁸M DHT or EtOH for 12 h and harvested for ChIP. Immunoprecipitation of sonicated chromatin solutions was conducted by overnight incubation at 4°C with anti-AR (Thermo), anti-BAP18, anti-MLL1, anti-hMOF, anti-Ash2L, anti-H3K27me3 (Abcam), anti-H3K4me3 (Abcam) or anti-H4K16ac (Millipore) antibody. Cross-linking was reversed at 65°C, and DNA fragments were extracted with phenol-chloroform and precipitated with ethanol. The purified DNA was dissolved in TE buffer and analyzed by regular PCR. Primer sequences of PSA-ARE I/II were as follows: forward 5′-GCCAAGACATCTATTTCAGGAGC-3′, and reverse 5′-CCCACACCCAGAGCTGTGGAAGG-3′. DNA fragments was analyzed by real-time PCR (RT-PCR) with SYBR Green dye. Results were expressed as percentage of input chromatin and were derived from a single experiment that is representative of at least two independent experiments.