Extended Data Figure 3: Physical association of AR with BRD4 and its disruption by BET bromodomain inhibitor.

a, LNCaP nuclear extract was fractionated on a Superose-6 column and AR, BRD4 and RNA Pol II were analysed by immunoblot analysis. b, c, Representative sensorgrams for AR–RNF2, RAS–BRD4(BD1–BD2) and RNF2–BRD4(BD1–BD2) interactions by an OctetRED biolayer interferometry. Real-time binding was measured by immobilizing biotinylated AR, RAS or RNF2 proteins separately on a streptavidin biosensor and subsequent interaction with varying concentrations of analyte proteins (RNF2 or BRD4(BD1–BD2)) individually. Immobilized RAS or RNF2 biosensors did not bind with BRD4, indicating that the AR–BRD4 interaction is specific. Representative sensorgrams from 4–6 independent experiment are shown. d– f, In vitro binding analysis of AR and indicated domains of BRD4. Equal amounts of in vitro translated full-length Halo–AR protein and GST–BRD4 domains were combined and immunoprecipitated using Halo beads followed by immunoblot analysis with anti-GST antibody. g, JQ1 disrupts the endogenous AR–BRD4 interaction. VCaP cells were treated with JQ1 for 6 h followed by immunoprecipitation and immunoblot analysis as in Fig. 2b.