Anti psa

Whole cell lysates were harvested and lysed in RIPA buffer containing the protease inhibitor cocktail (Sigma-Aldrich St. Louis, MO, USA). Western blot analysis was performed as described previously^{19 (link)}. The anti-PSA and anti-β-actin antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). The immunoreactive proteins were detected using horseradish peroxidase-conjugated anti-rabbit antibody (Cell Signaling Technology) and ImmunoStar (FUJIFILM Wako Pure Chemical).

Following the drug treatment, cells were lysed for 20 min on ice with buffer containing 50 mM Tris-HCl (pH 7.4), 5 mM EDTA 1% Nonidet P-40, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate, 50 mM NaF, 50 mM β-glycerolophosphate, 1 mM PMSF, 1 mM sodium orthovanadate, and a protease inhibitor cocktail (Roche Applied Science, Basel, Switzerland). Proteins (50 μg) were separated by SDS-PAGE and transferred to PVDF membranes. The following antibodies were used: anti-PSA (#5877 Cell Signaling Technology, Danvers, MA, USA), anti-AR (#5153S Cell Signaling Technology), anti-GAPDH-HRP conjugate (#8884S Cell Signaling Technology), anti-γH2AX (#05-636-I, Millipore, Burlington, MA, USA), #anti-p53 (DO-I, Santa Cruz Biotechnology, Dallas, TX, USA), anti-p21 (#2947 Cell Signaling Technology), anti-PARP (#9542 Cell Signaling Technology), and anti-β-actin (C4, Santa Cruz Biotechnology, Dallas, TX, USA). Immunoblots were developed using the Li-COR Odyssey imaging system, except for the anti-PSA, anti-AR, and anti-GAPDH-HRP conjugate, which were visualized using an enhanced chemiluminescence detection kit, the SuperSignal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, Waltham, MA, USA).

Total lysates prepared from subconfluent cells as described^{24 (link)} were subjected to immunoblot analysis using anti-AR (H-280, 1:100 dilution; Santa Cruz Biotechnology), anti-PSA (5365, 1:1000 dilution; Cell Signaling Technology, Danvers, MA, USA), anti-NKX3.1 (83700, 1:1000 dilution; Cell Signaling Technology), anti-β-actin (1:100,000 dilution; Sigma), anti-MUC1-C (HM-1630-P1ABX, 1:400 dilution; Thermo Fisher Scientific, Waltham, MA, USA), anti-EZH2 (5246, 1:1000 dilution; Cell Signaling Technology), anti-MYCN (9405, 1:1000 dilution; Cell Signaling Technology), anti-BRN2 (12137, 1:1000 dilution; Cell Signaling Technology), anti-MYC (ab32072, 1:1000 dilution; Abcam, Cambridge, MA), anti-SOX2 (3579, 1:1000 dilution; Cell Signaling Technology), anti-ASCL1 (GTX129189, 1:1000 dilution; GeneTex, Irvine, CA, USA), anti-AUROKA (ab1287, 1:4000 dilution; Abcam), anti-SYP (MA5-16402, 1:200 dilution; Thermo Fisher Scientific), anti-KLF4 (12173, 1:1000 dilution; Cell Signaling Technology) and anti-OCT4 (2750, 1:1000 dilution; Cell Signaling Technology).