Anti ar

The cell lysates were prepared from PCa cells treated with GTEE or vehicle (0.3% ethanol) using PRO-PREP Protein Extraction Solution (iNtRON technology, South Korea) with protease inhibitors added. The protein concentrations of the cell lysates were assayed using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). For Western blotting, 50 µg of protein extract was loaded into the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gel and transferred onto polyvinylidene difluoride (PVDF) membranes. After blocking by 5% non-fat milk in PBST (PBS with Tween-20) buffer for 1 h at room temperature, PVDF membranes were incubated with primary antibodies for overnight at 4 °C, followed by incubation with a horseradish peroxidase (HRP)-conjugated secondary antibody. The reactive signals were visualized using an Enhanced Chemiluminescence Kit (Amersham Biosciences, Arlington Heights, IL, USA). Subsequently, the reactive protein bands were scanned and quantified using ImageJ software. Primary antibodies were used as follows: anti-SREBP-1, anti-FASN, anti-AR (Santa Cruz Biotechnology, Dallas, TX, USA), anti-SREBP-2 (Abcam, Cambridge, MA, USA), anti-caspase 3 (Novus Biologicals, Littleton, CO, USA), anti-PARP (GeneTex, Irvine, CA, USA), and anti-β-actin (Millipore, Burlington, MA, USA).

MDA-MB-231 and DU145 xenografts were fixed in 10% formalin and embedded with paraffin. Embedded tissues were sectioned (10 µm) for immunohistochemistry experiments. Prior to immunostaining, paraffin sections were deparaffinized and rehydrated. Paraffin sections were first boiled in 10 mM citrate buffer (pH 6.0) to retrieve antigenicity. Sections were incubated in 3% H₂O₂ in PBS for 10 min at room temperature to quench endogenous peroxidase and incubated in 10% normal serum (prepared from the species in which the secondary antibodies were generated) for 1 h at room temperature to block the non-specific binding sites. Sections were incubated with the primary antibody for 1 hr at room temperature or overnight at 4 C. Antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA): anti-AR (catalog #sc-7305), and anti-ERα (catalog #sc-543). Following washes, sections were treated with secondary antibody for 1 h at room temperature. Then the Vectastain ABC kit (Vector Laboratories, Burlingame, CA) was used per manufacturers’ instructions. Peroxidase activity was visualized with 3,3’-diaminobenzidine. Negative controls were incubated with non-specific IgG.

Protein extracts were prepared in a cell lysis buffer and denatured at 95 °C for 10 min, separated on SDS-PAGE gels and transferred to nitrocellulose membranes (Bio-rad Laboratories, Hercules, CA, USA). After blocking in 5% non-fat milk in T-TBS, membranes were incubated overnight at 4 °C with primary antibodies in 0.5% BSA or 2% non-fat milk in T-TBS: anti-AR (1:1000 dilution; Santa Cruz Biotechnology, Dallas, TX, USA; #7305), anti-RUNX1 (1:1000; Cell Signaling Technology; #4334), anti-SOX4 (1:1000; Abcam #52043), anti-Tubulin (1:10,000; Sigma-Aldrich; #T5168), and anti-GAPDH (1:5000; Santa Cruz Biotechnology; #32233). Secondary antibody incubation was performed at room temperature for 1 h: anti-mouse (1:5000; Li-Cor #926-32213 or Li-Cor #926-68070) or anti-rabbit (1:5000; Li-Cor; Lincoln, NE, USA; #926-32210). Membranes were then scanned using the Odyssey Imaging System and analyzed with Image Studio Ver 5.2 software (Li-Cor). Quantifications are presented below each blot as the relative mean of all experiments performed plus/minus the standard deviation, normalized to the housekeeping. All experiments were performed at least three times.