Androgen receptor

The experimental methods for protein quantification and analysis were all adapted from our previous publication [14 (link)]. Briefly, approximately 30 mg of left ventricle tissue was excised from both groups and homogenized in cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) containing a protease cocktail (Sigma Life Science, Burlington, MA, USA) and PMSF. The homogenized samples were centrifuged at 15,000 rpm for 30 min at 4 °C, and supernatant was quantified for 50 μg of equivalent proteins using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Each sample was denatured and loaded into an SDS-PAGE precast gel (Bio-Rad Laboratories, Hercules, CA, USA). Blots were later blocked in 5% w/v nonfat dry milk for 1 h and probed in 1:5000 dilutions of primary antibodies for Kv1.4 (AB5926) and GAPDH (Millipore) antibodies, and 1:1000 dilutions of KChIP2 (Abcam, Cambridge, MA, USA), Kv4.2 (Millipore, Billerica, MA, USA), estrogen receptor beta (Thermo Fisher, CA, USA), androgen receptor (Abcam, Cambridge, MA, USA), and Kv1.5 antibody (Santa Cruz Biotechnology, Dallas, TX, USA). The band intensities from GAPDH were used to normalize the target proteins using ImageJ software.

LNCaP and C4–2 cells were treated with GPB730 and enzalutamide for 72 h. Cell lysates were prepared and western blot analysis was performed according to previous publication [21 (link),22 (link)]. Primary antibodies used were anti-STAT3 (#4904 Cell Signaling Technology), anti-pSTAT3-T705 (ab76315 Abcam), anti-pSTAT3-S727 (#9134 Cell Signaling Technology), c-myc (ab32072 Abcam), survivin (#2808 Cell Signaling Technology), PSA (sc7638 Santa Cruz) and androgen receptor (ab108341 Abcam). Beta-actin (#A5441 Sigma) was used as loading control. LNCaP cells stimulated with 50 ng/ml IL-6 for 30 min and DU145 cells were used as positive controls for pSTAT3-S727 and pSTAT3-T705 expression.