Subcellular Localization of AR Variants

Subcellular localization of AR proteins was analyzed by confocal fluorescence microscopy. The pTurboFP635-AR-V7 and pTurboFP635-AR^v567es plasmids were generated by cloning the cDNA fragments for AR-V7 and AR^v567es, respectively, into the pCMV-TurboFP635 vector. COS-7 cells were transfected with indicated plasmids and cultured in phenol red-free RPMI-1640 supplemented with 10% cs-FBS. At 40 hr after transfection, cells were pre-treated with or without 10 nM docetaxel for 6 hr, followed by treatment with or without 1 nM R1881 for 4 hr. COS-7 cells were subsequently fixed with 2% paraformaldehyde, and the nuclei were stained with 2.5 μM DRAQ5 (Cell Signaling). Confocal images were obtained by using a Leica TCS SP2 system with a 63X oil-immersion objective on a Z-stage, and an average of 6 fields with ∼10 cells per field were captured for each group. Data quantitation was performed as described [44 (link)].