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Figure 5 | BMC Biochemistry

Figure 5

From: Ski-interacting protein (SKIP) interacts with androgen receptor in the nucleus and modulates androgen-dependent transcription

Figure 5

AR interacts with SKIP after nuclear translocation on SKIP-positive nuclear speckles. (A,B) Donor dequenching and (C-E) sensitised acceptor emission FRET imaging data of AR-ECFP and EYFP-SKIP co-expressed in BHK cells. (A) BHK cells were transiently transfected with AR-ECFP and EYFP-SKIP. Before being fixed with 4% PFA, cells were stimulated for 2.5 min with 10 nM DHT at room temperature. The acceptor was then bleached and the increase of the donor-fluorescence was monitored in the donor channel, using confocal imaging. (B) The average FRET efficiency of AR-ECFP/EYFP-SKIP (8 images, 24 speckles), E%=57±3% (mean±SEM) was significantly higher (P<0.001, 2-tailed Student’s t-test), than that of the control FRET-pair, ECFP/EYFP-SKIP, E%=11±2% (mean±SEM) (3 images, 9 speckles) on EYFP-SKIP positive nuclear speckles. This is in line with a strong interaction between AR and SKIP on these structures. (C) Representative time series of serum starved BHK cells, transiently transfected with AR-ECFP and EYFP-SKIP that were stimulated with 10 nM DHT (+DHT). Using confocal imaging, nuclear translocation of AR-ECFP and FRET-changes (FRET2) were measured over time. Time points are shown in FRET2 images. The approximate boundary of the nucleus is indicated in the first AR-ECFP image. (D) The nuclear translocation index of data in C was calculated using the largest speckle (arrow in first image) and a region in the cytoplasm (asterisk) (apparent translocation half-time, τ0.5=213±9 s). (E) The change of the FRET2 signal (FRET2) and the FRET2 signal normalized with the unprocessed acceptor signal (FRET2/Ac) were averaged over the whole nucleus for each time point. Both FRET signals reached their maximum at ~ 129 s after DHT addition, corresponding to ~ 32% translocation. Subsequently the FRET signal decreased, as the fluorescence of EYFP-SKIP disappeared.

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