(A) Schematic of the TALEN approach to knockout Bag-1L expression. The TALEN target sequences are highlighted in grey and the start codon (CTG) of Bag-1L is highlighted in yellow. Note, the start codons for the other Bag-1 isoforms remain intact. The resulting coding sequence, which is missing the Bag-1L start codon and flanking regions, is shown in red. (B) Western blot of nuclear extracts from hormone-deprived control (Ctr) and Bag-1L KO (KO) cells treated with vehicle (ETOH) or 10 nM DHT for 4 hr. Protein levels of Bag-1 isoforms (Bag-1L, −1S and −1, but not −1M) and AR are shown. The expression of the nuclear protein Lamin B1 was used for equal protein loading. (C) Western blot of Bag-1L and AR levels in control, Bag-1L KO and Bag-1L KO rescue cell lines, as indicated. β-actin was probed to ensure equal protein loading. (D) Proliferation assay of indicated cell lines grown in complete media and counting on days 0 and 5. Data are the averages of three independent experiments ± SEM, normalized to day 0. p-values were calculated using standard t test; *p≤0.05; ***p≤0.001; ****p≤0.0001. (E) Venn diagram of AR cistromes from replicate ChIP-seq experiments in hormone-depleted control (red) and Bag-1L KO (blue) cells treated for 4 hr with DHT. The union of binding sites is indicated. (F) Signal profiles and heatmaps of AR ChIP-seq data (centered on the AR peaks) from indicated cell lines. Signals are shown for sites with an AR peak in one or more cell lines. Cells were starved of hormone and treated with 10 nM DHT for 4 hr prior to the experiment. (G) Top, fluorescence images of CFP-AR-YFP-expressing hormone-depleted control or Bag-1L KO cells, treated with ETOH or 10 nM DHT for 2 hr. The FRET channel, which is corrected by 17% of ICFP, is shown in red, the raw CFP in blue, and the YFP, under direct excitation at 488 nm, in yellow. Results are from two independent experiments with more than 10 images. Scale bar: 10 µm. A schematic representation of the CFP-AR-YFP construct is shown below. Bottom, Quantification of CFP-AR-YPF FRET. IFRET/(IFRET +IDonor) ratios in the nucleus are shown. Data is the result of readings from more than 25 cells per sample and is presented as mean ±SD. ****p≤0.0001.