Anti ar

Enzalutamide (Cat# A10562) was purchased from Adooq Bioscience, Irvine, CA, USA, and all other reagents purchased were of GLP grade. Antibodies including Anti-AR (Cat# 5153), Anti-AR-v7 (Cat# 19672), anti-POU5F1 (OCT4) (Cat# 2750S) were purchased from Cell Signaling Technologies, Beverly, MA, USA. The anti-ALDH1 (Cat# SC-166362), anti-SOX2 (Cat# SC-365823), anti-α-GAPDH (Cat# SC-47724), goat anti-mouse IgG-HRP (Cat# SC-2005), bovine anti-goat IgG-HRP (Cat# SC-2350), and goat anti-rabbit IgG-HRP (Cat# SC-2004) antibodies were purchased from Santa Cruz Biotechnology, Dallas, TX, USA.

ChIP experiments were carried out according to the standard protocol (Merck KGaA, Darmstadt, Germany). LNCaP cells were plated in 15-cm tissue culture plates and transfected with siCREB3L4 siRNA, combined with 10 nM R1881 treatment. Cell lysates were then incubated for 15 hr at 4 °C with 5-μg anti-AR (#5153, Cell Signaling) or normal rabbit IgG (sc-2027, SantaCruz Biotechnology) antibodies. After reversal of crosslinking, immunoprecipitated DNA was quantified by qPCR. All reactions were normalized relative to total input to account for chromatin sample preparation differences (ΔC_t), determined as the difference between the α-AR IP sample (ΔC_{t [AR]}) and α-IgG IP sample (ΔC_{t [IgG]}) for fold enrichment. Relative expression was determined by the method ΔΔC_{t [AR - IgG]} = ΔC_{t [AR]} - ΔC_{t [IgG]}, and fold-change in occupancy = 2^{(−ΔΔCt[AR-IgG])}30 (link). The primer used for PCR of the AR promoter binding region (distal ARE) of the PSA gene is described in ref. 31 (link). Primers sequence for PCR of four putative AREs were as follows: ARE1-f, 5′-AACCTGGATTCTGGTCCAAGTTCTA-3′; ARE1-r, 5′-AACTCCTGACCTCGTCATCTGC-3′; ARE2-f, 5′-TCAAATCATCTCTGCACATACA-3′; ARE2-r, AACCAAGATTTGGATGCTTCAG-3′; ARE3-f, 5′-GGAGCTTGCAGTGAGCCGAGATC-3′; ARE3-r, 5′-ACTGCTACTAATTATCCTTATGAAG-3′; ARE4-f, 5′-TGCATGGAACCGTGATCGCACCAC-3′; ARE4-r, 5′-AAGCTGAAGACTTAGGTTTCGGAG-3′.

Homogenization was performed using the lysis buffer (Beyotime, Wuhan, China). Bradford assay (Bio‐Rad, Roseville, CA, USA) was used for protein quantification. SDS/PAGE was used to study these proteins, which were isolated using 8–15% polyacrylamide gels (Bio‐Rad) and transferred to polyvinylidene difluoride membranes (Millipore, Franklin, MA, USA). After blocking, the membranes were incubated overnight (4 °C) in the presence of particular primary antibodies (anti‐IGFR, anti‐phospho‐IGFR, anti‐AKT, anti‐phospho‐AKT, anti‐AR, anti‐IRS, anti‐phospho‐IRS, anti‐β‐actin, and anti‐LKB1; Cell Signaling Technology, Franklin, MA, USA). Secondary antibodies were subsequently supplemented. Enhanced chemiluminescence plus detection reagent (Pierce, Braketown, IL, USA) was used to measure the bands, which were further evaluated using the Omega 16ic Imaging System (Ultra‐Lum, Roseville, CA, USA).

IU1 (S7134), enzalutamide (S1250) and MK2206 (S1078) were obtained from Selleckchem (Houston, TX, USA). DMSO was used to dissolve these inhibitors and the inhibitors were stored at − 20 °C. USP14 (sc-76,817) siRNA was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). MTS (catalog no. G111) was obtained through Promega Corporation (Madison, WI, USA). Annexin V-FITC/PI apoptosis detection kits of Keygen Company (Nanjing, China)(KGA107) were purchased. Cell Lysis Buffer (#9803) was from Cell Signaling Technology (MA, USA) and stored at − 20 °C. Anti-GAPDH (MB001) was obtained from Bioworld Technology (St.Louis Park, MN, USA). The other antibodies were from Cell Signaling Technology (MA, USA): anti-PARP (#9542), anti-caspase 3 (#9668), anti-cleaved caspase 3 (#9661), anti-caspase 8 (#9746), anti-cleaved caspase 8 (#9496), anti-cleaved caspase 9 (#9501), anti-Bax (#5023), anti-CDK4(#12790), anti-P27(#3686), anti-caspase 9 (#9508), anti-CDK2 (#2546), anti-CyclinD1(#2922), anti-total-AKT (#9272), anti-AR (#5153), anti-USP14 (#11931), anti-Bcl-2 (#15071), anti-GSK3β (#12456), anti-p-GSK3β (Ser9)(#9323), anti-phospho-AKT (Ser473)(#4060), anti-β-catenin (#8480), anti-EGFR (#4267), anti-IGF1R (#9750).

A custom-built kinase inhibitor library (partial class of kinase inhibitors selected by authors), SNS-032 (#S1145), Dinaciclib (#S2768), Cycloheximide (#S7418), MG132 (#S2619), Bafilomycin A1 (#S1413), bortezomib (#S1013), and enzalutamide (#S1250) were obtained from Selleckchem (Houston, TX). Antibodies: anti-USP1 (#8033), anti-SIX1 (#16960), anti-Cyclin D1 (#55506), anti-p21 (#2947), anti-p27 (#3686), anti-cleaved Caspase 3 (#9661), anti-FLAG-tag (#8146), anti-Ki-67 (#9449), anti-AR (#5153), and anti-AR-V7 (#19672) were from Cell Signaling Technology (Beverly, MA); anti-GAPDH (#ab181602) were from Abcam (Cambridge, MA).

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).

Cell lysates were prepared and processed as previously described [43 (link)]. Membranes were incubated overnight with the following primary antibodies: anti-IGF-1Rβ, anti-IRβ, anti-GAPDH, anti-LAMIN B, anti-ERG-1/2/3 (Santa Cruz Biotechnology); anti-AR (Cell Signaling Technology); anti-rabbit or anti-mouse antibodies conjugated to horseradish peroxidase (GE Healthcare) were used as secondary antibodies.