Sc 816

Bone tissue sections were rehydrated through xylene and graded ethanol, incubated in the proteinase K (20 μg/ml) for 10 min and then sections were blocked for endogenous peroxidase with 3% hydrogen peroxide (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min in room temperature. Sections were permeabilized and blocked in 10% goat serum for 1 hr. The primary antibodies were anti-collagen I (1:600, ab138492 and ab21286, Abcam, Cambridge, MA, USA) diluted in the 5% goat serum and incubated at 4°C overnight. Sections were then incubated with a biotinylated goat anti-rabbit antibody (BD Pharmingen, San Diego, CA, USA). For detection, Streptavidin-Horseradish Peroxidase and DAB Substrate Kit (BD Pharmingen, San Diego, CA, USA) were used and the counterstain was done with hematoxylin. For osteocalcin (AbD Serotec 7060–1815, 1:1200 dilution), androgen receptor (Santa Cruz, sc-816, 1: 200) and ERβ (Santa Cruz, sc-8974) antigen retrieval was done with 0.5% trypsin at 37°C for 30 min and for estrogen receptor alpha (Santa Cruz 8002, 1:150), antigen retrieval was done in 10 mM sodium citrate, pH 6, at 95°C for 15 min.

The primary antibodies used and their subsequent dilutions were: anti-P16 (rabbit, 1:1000, SC-1207, Santa Cruz), anti-human AR (mouse, 1:250, sc-7305, Santa Cruz Biotechnology), anti-CK5 (rabbit, 2400, PRB-160P, Covance), anti-CK8 (mouse, 1:2000, MMS-162P, Covance), anti-p63 (mouse, 1:2000, sc-8431, Santa Cruz), anti Ki67 (mouse, 1:1000, NCL-ki67, Novacastra), anti-E-cadherin (mouse, 1:200, Cat. No. c20820, BD Transduction Laboratoriesc, Sparks, MD, United States), anti-mouse/ human androgen receptor (rabbit, 1:250, sc-816, Santa Cruz), anti-synaptophysin (rabbit, 1:100, Cat. No. 18–0130, Invitrogen), anti-SPP1 (rabbit, 1:200, Cat. No. 91655, Abcam, Cambridge, MA, USA), CD44 (Rat, 1:50, sc-18849, Santa Cruz) and anti-Vimentin (Chicken, 1:2000, Cat. No. 919101, Biolegend). The biotinylated anti-rabbit or anti-mouse secondary antibody (BA-1000 or BA-9200, Vector Laboratories), or anti-rabbit or anti-mouse conjugated to AlexaFluor488 or to AlexaFluor594 (Molecular Probes) secondary antibody that were used for IHC or IF staining, respectively.

Antibodies against AR (sc-816), prostate-specific antigen (PSA; sc-7638) and FKBP5 (sc-11514) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). anti-HER2 (#2165), anti-phosphorylated Y-box binding protein-1^Ser102 (p-YB-1; #2900), anti-α-tubulin (#2125), anti-Akt (#9272), anti-phosphorylated Akt^Ser473 (p-Akt; #4060), anti-PARP (#9542), and anti-cleaved PARP (#9541) antibodies were obtained from Cell Signaling (Danvers, MA). Antibodies against Lamin B1 (ab16048) were purchased from Abcam (Cambridge, MA). Anti-YB-1 (2397-1) antibodies were obtained from Epitomics (Burlingame, CA). Anti-β-actin (A3854) antibodies were purchased from BD Biosciences (Franklin Lakes, NJ) and Sigma (St Louis, MO).

The RIME technique was performed as described previously [13 (link)]. Briefly, cells (MDA-MB-453, MFM-223, ZR-75-1, and T-47D) were seeded at approximately 80% confluence in their appropriate growth medium and cultured for 48 h, then cross-linked in warm medium containing 1% formaldehyde for 7 min, quenched with 0.2 M Glycine, chromatin isolated and sonicated then subjected to immunoprecipitation using magnetic beads pre-bound with 10 μg of AR antibody (Santa Cruz Biotechnology Cat# sc-816, RRID:AB_1563391). An on-bead peptide digestion was performed, and a 2–5 μL aliquot of diluted peptide mixture was analyzed by Nano-LC–MS/MS. Peptides were identified using Proteome Discoverer (v1.4) (RRID:SCR_014477) and Mascot (RRID:SCR_014322) and/or SEQUEST (ProteinProphet (RRID:SCR_000286)) search engines as described in [13 (link)]. Only those interacting proteins that were identified in 3 of 3 independent biological replicates were considered for further analysis. Additional filtering was achieved by excluding non-specific interactions that appeared in > 1 of the 3 replicates of matching IgG negative controls.