Skip to main content
. 2022 Jul 18;11:e73396. doi: 10.7554/eLife.73396

Figure 6. DNA-binding mutation increases the intranuclear mobility and abrogates the transcriptional activity of AR-fl and AR-V7.

(A, B) FRAP was performed in PC3 cells transiently expressing GFP-AR-fl or GFP-AR-V7 or their respective DBD mutants (A573D). Kinetics of protein recovery after photobleaching are graphically displayed and their half-time of recovery, is obtained for each condition, n>14. (C) PC3 cells stably expressing ARE-mCherry reporter were transfected with indicated plasmids, in the presence or absence of ligand (10 nM R1881). Representative images of each condition are shown. (D) quantitation of mCherry fluorescence intensity in single cells (n>17). (E, F) The binding of (E) AR-fl or (F) AR-V7 on the enhancer of PSA or FBKP5 in 22RV1 was analyzed by ChIP-QPCR assay. Cells in charcoal stripped media were treated with vehicle or 10 nM R1881 for 24 hr. (G) Immunoblot for AR-fl and AR-V7 following subcellular fractionation CE, cytosolic extract; NE, nuclear extract, CB, chromatin-bound nuclear extract. Histone H3 and β-tubulin were used as controls for the fractionation. Data represent mean ± SEM, p value (**p<0.01, ****p<0.0001) was obtained using unpaired two-tailed t-test. Experiments were repeated at least twice.

Figure 6—source data 1. DNA-binding mutation increases the intranuclear mobility and abrogates the transcriptional activity of AR-fl and AR-V7.

Figure 6.

Figure 6—figure supplement 1. DBD mutation increases the intranuclear mobility of liganded-AR-fl and AR-V7.

Figure 6—figure supplement 1.

(A) FRAP was performed in the nuclei of PC3 cells following transient expression of GFP-AR-fl or GFP-AR-fl A573D (in the presence of 10 nM R1881) or GFP-AR-V7 or GFP-AR-fl A573D. Representative images of cells at the indicated time points are shown. Arrow heads show the photo-bleaching area. Scale bar, 10 µm. Experiments were repeated at least twice.