Ab198394

Unless otherwise noted, all primers and restriction enzymes were purchased from Takara Biotechnology (catalog number Xho I 1635, Sma I 1629). All primary antibodies were purchased from Abcam: Anti-HIV TAT (N3), ab63957; EVs Anti-CD63[EPR5702], ab134045; Anti-ALIX [3A9], ab117600; Anti-Androgen Receptor (AR-V7 specific) [EPR15656], ab198394. The secondary antibody was purchased from Odyssey (Goat anti-Mouse antibody, IRDye 680). Lipofectamine 3000 Transfection Reagent was purchased from Life Technology (L3000008). Plasmid pET-44bC vector was purchased from Novagen (71123–3). T4 DNA ligase was purchased from New England BioLabs (M0202S). His GraviTrap Kit for protein purification was purchased from GE Healthcare (11–0036-90 AA). TRIzol was purchased from Life Technology (15596026). Reverse transcription kit and real-time quantitative PCR kit were purchased from Takara Biotechnology (RR036A and RR820A). Cell Counting Kit-8 (CCK8) was purchased from Dojindo (CK04). Negative control siRNA (siRNA-NC), Cy5-labeled control siRNA (Cy5-siRNA), FAM-labeled control siRNA (FAM-siRNA), therapeutic siRNAs targeting FLOH1, NKX3, DHRS7 genes, and the negative control siRNA were synthesized by RiboBio Co., Ltd. All other reagents and chemicals were of analytical grade and used without further purification.

The slides were deparaffinized, hydrated, blocked with 30% H₂O₂ in distilled water to eliminate endogenous peroxidase activity, and incubated with boiling buffered citrate. The tissue expression of AR was determined using a primary antibody against AR (ab198394, Abcam Inc., Cambridge, MA, USA) at a 1:250 dilution and the Vectastain Elite ABC Kit (Vector Laboratories, Burlingame, CA) according to the manufacturer’s instructions, followed by visualization using FAST DAB Peroxidase Substrate (Sigma, St Louis, MO, USA). The slides were observed and photographed under a light microscope. The negative controls were obtained after exchanging primary antibodies with PBS.

Cell lysates were prepared using RIPA Buffer (Boston BioProducts) supplemented with protease inhibitors (Sigma) and phosphatase inhibitor (Active Motif). For immuoprecipitation experiments, cell lysates were incubated with desired antibodies in Tris buffered saline buffer at 4 °C overnight, then protein G agarose (Beyotime Biotechnology) was added to each sample for another 2 h incubation, followed by five times wash with PBS solution and boiling for IB. To avoid the interference from denatured IgG heavy chain, VeriBlot IP Detection Reagent (ab131366, Abcam) was used for IP detection. The antibodies against ARV7 (ab198394), Bcl-2 (ab59348), MCL-1 (ab28147) and Bim (ab15184) were obtained from Abcam while antibodies against cleaved-PARP (5625), Bid (2002), UBE2C (14234), p-H3(ser10) (9701), BubR1 (4116), and Cdc20 (14866) were purchased from Cell Signaling Technology, and the antibodies against GFP (sc-9996), p53 (sc-126), Mad-2 (sc-47747), and ubiquitin (sc-8017) were obtained from Santa Cruz. Proteintech is the provider of antibodies against β-actin.

For Western blot analysis, proteins isolated from the control cell samples (LNCaP and PC3) and from the experimental cell samples were collected after incubation with 5.0 µM erastin for 24 h. In addition, proteins isolated from the control cell samples (LNCaP and VCaP) and from the experimental cell samples were collected after incubation with 10.0 µM enzalutamide for 48 h. Each protein from these samples was separated in a 10% sodium dodecyl sulfate polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. These membranes were probed with primary antibodies against SLC7A11 (1:250, ab307601, Abcam, USA), TMEFF2 (1:500, ab133562, Abcam, USA), AR (1:500, ab198394, Abcam, USA), and β-tubulin (1:2000, 10094-1-AP, ProteinTech, China). Subsequently, the membranes and the corresponding secondary antibody (ProteinTech, China) were incubated at room temperature for 1 h before being photographed.

The samples were homogenized in ice-cold RIPA buffer (25 mM Tris/HCl (pH 7.6), 150 mM NaCl, 1% sodium deoxycholate, 1% Nonidet-P40, 0.1% SDS, and 0.05 mM PMSF). The protein concentration was quantified using a BCA protein assay (Thermo Fisher Scientific, Rockford, USA). Equal amounts of protein (40 μg/lane) were subjected to 8% SDS-PAGE and subsequently transferred to polyvinylidene di-fluoride (PVDF) (Millipore, Bedford, MA) membranes. After blocking in 5% fat-free dry milk, the membranes were incubated overnight at 4 °C with an anti-AR antibody (ab198394, Abcam Inc., Cambridge, MA, USA) diluted 1:1000. After washing, the membrane was incubated with peroxidase-linked goat anti-rabbit IgG (1:5000, BS13278, Bioworld Technology Inc., Louis Park, MN) for 2 h. Bound antibodies were detected using an ECL detection system (Vazyme Biotech, China). The immunoreactive bands were quantified using Quantity One software (Bio-Rad Laboratories). The validity of AR antibody in P. sinensis has been detected by negative and positive control analysis.