Ar fl antibody

22Rv1 and C4–2 cells were plated in hormone-deficient media for 72 hours. Following which, cells were treated with vehicle (0nM) or CCS1477 500nM for 5 hours, followed by the addition of 10nM dihydrotestosterone for 3 hours. Chromatin immunoprecipitation (ChIP) was performed as previously described (61 (link)). Briefly, cells were cross-linked with 1% fresh formaldehyde for 10 minutes at room temperature. Chromatin was sheared to 200–700bp using Diagenode Ultrasonicator for 30 cycles. Lysates were incubated with CBP or p300 custom antibody, or AR-FL antibody (Millipore, 06–680). ChIP DNA was extracted via phenol-chloroform method and qPCR was performed using PowerUP SYBR (ThermoFisher). Genes selected for ChIP-qPCR were prioritized based on statistically significant transcriptional downregulation after CCS1477 treatment (1.5 fold change; p=<0.05) and known AR regulation: KLK3, TMPRSS2, FKBP5, ANKRD30B, CHRNA2 (with TFF1 and TGFA representing known CBP binding sites) (48 (link), 49 (link)). Primers were designed at AR target genes using previously described AR binding sites and are listed in Supplementary Table 15 (62 (link)–64 (link)).

ChIP assays were performed exactly as previously described (71 (link)). Cell lysates were incubated with 5 μg AR-FL antibody (Millipore #06–680), 5 μg AR-V7 antibody (Cell Signaling #68492) or 5 μg rabbit anti-mouse Igg (Santa Cruz sc2027) respectively. The AR and AR-V7 recruitment were measured by qPCR using the primers and probes listed in Supplemental Table 1.