(A) Click-chemistry experiment. (B) EPI probes used for Click-chemistry. (C) LNCaP cells treated with EPI-046, EPI-047, and EPI-051 were lysed prior to Click-chemistry. Biotin-labeled probes covalently bound to cellular proteins were detected using an antibody against biotin (left), and AR protein was detected using an antibody against AR (middle). β-actin is a loading control (right). Dotted red outlines denote AR and β-actin bands, as indicated. (D) Western blot analysis of biotin-labeled probes covalently bound to cellular proteins from LNCaP cells treated with EPI-054 before (right) or after Click-chemistry and streptavidin enrichment (left). Gels were probed using anti-biotin or anti-AR antibodies, as indicated. Arrows denote bands corresponding to AR (110 kDa). (E) LNCaP cells treated with EPI chiral probes were lysed prior to Click-chemistry, enriched using streptavidin, and probed using anti-biotin antibody (top). AR levels prior to Click-chemistry were detected using anti-AR441 antibody (input; bottom). (F) Cells transfected with FLAG-AR NTD or vector were treated with EPI analogs. Proteins were detected using anti-biotin, anti-AR NTD, or anti-FLAG antibodies following Click-chemistry and streptavidin enrichment (top) or cell lysates before Click-chemistry (input; middle and bottom). Asterisks denote proteins that were also detected with anti-biotin antibody in DMSO samples not treated with EPI-probes. Lanes in D–F were run on same gel but were noncontiguous (black lines).