Rabbit polyclonal anti ar n 20

Western immunoblots were performed using the prepared lysates from assays as described above. Total cellular protein concentrations were determined by Coomassie Plus (Bradford) Protein Assay (Thermo Fisher Scientific Inc., Rockford, IL). Equivalent amounts of protein were loaded for each sample on 10–20% Criterion gels (BioRad), proteins were separated by electrophoresis, and proteins were transferred to Immobilon PVDF membranes (Millipore) following standard procedures. The following primary antibodies were used to detect proteins of interest: Rabbit polyclonal anti-β-catenin (Millipore), mouse monoclonal anti-FKBP52 (Hi52D, epitope in the FK1 domain) [34 (link)], rabbit polyclonal anti-AR (N-20; Santa Cruz Biotechnology), and mouse anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Biodesign International) as a loading control. The secondary antibodies used included AP-conjugated goat anti-rabbit and anti-mouse IgG secondary antibodies (Southern Biotechnology). The ImmunStar Alkaline Phosphatase Substrate (BioRad) was applied prior to exposing to X-ray films for development and detection of antibodies.

Dihydrotestosterone (DHT) and anti-androgen bicalutamide were purchased from Sigma-Aldrich Co., LLC. (St. Louis, MO, USA). Recombinant human EGF, FGF-2, FGF-7, FGF-10, hepatocyte growth factor (HGF), IGF-1, transforming growth factor (TGF) β1, vascular endothelial growth factor (VEGF), and IL-6 were purchased from PeproTech, Inc. (Rocky Hill, NJ, USA). Rabbit polyclonal anti-PSA and mouse monoclonal anti-neuron-specific enolase (NSE; BBS/NC/V1-H14) antibodies were purchased from Dako Cytomation (Copenhagen, Denmark). Rabbit polyclonal anti-AR (N-20) and anti-EGFR (1005) antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Rabbit polyclonal anti-CD31 and anti-Ki-67 and rabbit monoclonal anti-AR splice variant 7 (AR-V7; EPR15656) antibodies were purchased from Abcam Inc. (Cambridge, MA, USA). Rabbit monoclonal anti-phospho-STAT3 (Tyr705) (D3A7), anti-STAT3 (D3Z2G), anti-phospho-Akt (Ser473) (D9E), and anti-Akt (pan) (C67E7) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). Rabbit polyclonal anti-p44/42 MAPK and mouse monoclonal anti-phospho-p44/42 MAPK (Thr202/Tyr204) (E10) antibodies were purchased from Cell Signaling Technology, Inc. Mouse monoclonal anti-β-actin (AC-15) antibodies were purchased from Sigma-Aldrich Co., LLC.