Anti ar

The procedure was described previously [36] (link). Immunoreactive bands were quantitated by densitometry and normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). The following antibodies were used: anti-GAPDH (Millipore), anti-AR (Millipore), and anti-AR-V7 (Precision Antibody).

Protein extracts were prepared as previously described [28 (link)]. Briefly, cells were lysed in [30 mM Tris pH 7.5; 100 mM NaCl; 10 mM MgCl2; 1% Triton X-100; 1 mM DTT; 0,5 mM Na3VO4; protease inhibitor cocktail (Sigma Aldrich)], briefly sonicated, incubated on ice for 20 min and centrifuged for 20 min at 12,000 g at 4 °C. Protein extracts were then analysed by Western Blot using the following primary antibodies: rabbit anti-HER3/ErbB3 (D22C5) (mAb#12,708, Cell Signaling), anti- AR (06–680, Millipore), anti-PCF11 (A303-706A, Bethyl Laboratories), anti-CSTF3 (A301-094, Bethyl Laboratories), anti-RBM39 (HPA001591, Atlas Antibodies), anti-H3 (Ab1791, Abcam), anti-H3K27me3 (mAb#9733, Cell Signaling); mouse anti-HSP90 (sc-13119, Santa Cruz), anti-ACTIN (sc-47778 Santa Cruz), anti-EZH2 (AC22) (mAb#3147, Cell Signaling), anti-MDM4 (clone 7A8, 04–1556, Sigma Aldrich). Anti-rabbit, anti-mouse (GE Healthcare) HRP-linked secondary antibodies were all used at 1:5000 dilution and ECL signal developed using Clarity Western ECL Substrates (Biorad) or Amersham ECL Select™. Densitometric analyses were performed using Alliance system software (UVITEC, Cambridge).

LNCaP, 22Rv1, and COS-7 cells were obtained from American Type Culture Collection. With the exception of drug-resistant lines, cells used in this study were within 20 passages (∼3 months of non-continuous culturing). All cell lines were tested and authenticated by the method of short tandem repeat profiling. Docetaxel, cabazitaxel, and paclitaxel were purchased from Selleck Chemicals (Houston, TX). Nocodazole was from Sigma Aldrich, and KX-01 was provided by Kinex Pharmaceuticals. The following antibodies were used in Western blot analysis: anti-GAPDH, anti-AR (N-terminus-directed, PG-21; Millipore), anti-importin β1, anti-β-actin (Santa Cruz), anti-p53 (Calbiochem), anti-histone H3 (Cell Signaling), and anti-AR-V7 (Precision Antibody).

Protein–DNA complexes were pre-fixed in solution A (50 mM Hepes-KOH, 100 nM NaCl, 1 nM EDTA, 0.5 EGTA) with 2 mM disuccinimidyl glitarate (DSG) (CovaChem) for 35 min at room temperature, followed by fixation with 1% formaldehyde for 10 min and subsequently quenched with glycine. After three PBS washes, cells were collected in lysis buffer containing proteinase inhibitors (10 nM Tris-HCl, 100 mM NaCL, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxycholate, 0.5% N-lauroylsarcosine) for nuclei extraction, followed by sonication for at least 10 cycles of 30 s on and 30 s off using a Diagenode Bioruptor Pico. Size of the DNA segments was evaluated on agarose gel. Lysate was then incubated with the specific antibodies overnight. Antibodies used were: Anti-AR (Millipore, 06-680) (7 µg per sample) and anti-H3K27ac (Active Motif, 39133) (5 µg per sample). After incubation, reverse crosslinking was performed at 65 °C overnight. DNA was then treated with 1 mg ml⁻¹ RNAseA for 1 h at 37 °C followed by treatment with proteinase K for 2 h at 55 °C. DNA was then collected with phenol-chloroform protocol and submitted for sequencing.