| . | . | FRAP t1/2 (s)f . | |||||
|---|---|---|---|---|---|---|---|
| . | Kinetic component . | NB . | UBD . | Anchor . | Reader . | Reader + FVP . | Reader + C1 . |
| Reader1.0 Nucleoplasma | Fast | 0.6 ± 0.3 (100%)c | 0.7 ± 0.4 (100%)c | 0.7 ± 0.3 (80.5%)d | 1.1 ± 0.5 (72.6%)d | 0.9 ± 0.5 (68.4%)d | 0.8 ± 0.4 (79.1%)d |
| Slow | 10.2 ± 7.8 (19.5%)d | 11.0 ± 7.2 (27.4%)d | 6.5 ± 4.0 (31.6%)d | 8.3 ± 7.2 (20.9%)d | |||
| Reader2.0 Nucleoplasma | Fast | 1.9 ± 1.2 (43.1%)d | 1.6 ± 0.9 (60.8%)d | 1.5 ± 0.5 (74.9%)d | |||
| Slow | 20.2 ± 6.8 (56.9%)d | 11.0 ± 5.9 (39.2%)d | 14.6 ± 9.5 (25.1%)d | ||||
| Reader2.1 Nucleoplasma | Fast | 0.7 ± 0.4 (100%)c | 0.6 ± 0.3 (100%)c | 1.4 ± 0.7 (69.3%)d | 1.3 ± 0.9 (26.1%)d | 1.5 ± 0.6 (59.2%)d | 1.2 ± 0.5 (74.3%)d |
| Slow | 10.2 ± 6.6 (30.7%)d | 31.6 ± 6.1 (73.9%)d | 15.9 ± 5.5 (40.8%)d | 10.6 ± 6.9 (25.7%)d | |||
| Reader1.0 Nucleoplasmb | Fast | 0.6 ± 1.2 (69.6%)e | |||||
| Slow | 4.5 ± 9.8 (30.4%)e | ||||||
| Reader1.0 DSBsb | Fast | 0.9 ± 1.7 (16.4%)e | |||||
| Slow | 15.1 ± 2.9 (83.6%)e | ||||||
| . | . | FRAP t1/2 (s)f . | |||||
|---|---|---|---|---|---|---|---|
| . | Kinetic component . | NB . | UBD . | Anchor . | Reader . | Reader + FVP . | Reader + C1 . |
| Reader1.0 Nucleoplasma | Fast | 0.6 ± 0.3 (100%)c | 0.7 ± 0.4 (100%)c | 0.7 ± 0.3 (80.5%)d | 1.1 ± 0.5 (72.6%)d | 0.9 ± 0.5 (68.4%)d | 0.8 ± 0.4 (79.1%)d |
| Slow | 10.2 ± 7.8 (19.5%)d | 11.0 ± 7.2 (27.4%)d | 6.5 ± 4.0 (31.6%)d | 8.3 ± 7.2 (20.9%)d | |||
| Reader2.0 Nucleoplasma | Fast | 1.9 ± 1.2 (43.1%)d | 1.6 ± 0.9 (60.8%)d | 1.5 ± 0.5 (74.9%)d | |||
| Slow | 20.2 ± 6.8 (56.9%)d | 11.0 ± 5.9 (39.2%)d | 14.6 ± 9.5 (25.1%)d | ||||
| Reader2.1 Nucleoplasma | Fast | 0.7 ± 0.4 (100%)c | 0.6 ± 0.3 (100%)c | 1.4 ± 0.7 (69.3%)d | 1.3 ± 0.9 (26.1%)d | 1.5 ± 0.6 (59.2%)d | 1.2 ± 0.5 (74.3%)d |
| Slow | 10.2 ± 6.6 (30.7%)d | 31.6 ± 6.1 (73.9%)d | 15.9 ± 5.5 (40.8%)d | 10.6 ± 6.9 (25.7%)d | |||
| Reader1.0 Nucleoplasmb | Fast | 0.6 ± 1.2 (69.6%)e | |||||
| Slow | 4.5 ± 9.8 (30.4%)e | ||||||
| Reader1.0 DSBsb | Fast | 0.9 ± 1.7 (16.4%)e | |||||
| Slow | 15.1 ± 2.9 (83.6%)e | ||||||
FRAP of Reader1.0/2.0/2.1-eGFP transiently transfected in U-2 OS cells; n ≥ 6 cells per condition (two to five measurements per cell); photobleaching of circular ROIs in the nucleus (diameter = 30 pixels; 1 pixel = 0.09 × 0.09 µm).
FRAP of Reader1.0-eGFP stably expressed in U-2 OS cells; n = 6 cells (two measurements per cell in both the nucleoplasm and at laser-induced DSBs); rectangular ROI (0.75 µm2). Note that the ROI in these experiments is ∼7.5-fold smaller than the ROI used in the FRAP measurements of Reader1.0/2.0/2.1 in the nucleoplasm. The differences observed for the slow FRAP t1/2 of Reader1.0 in the nucleoplasm suggest that sensor’s diffusion rate has an impact in its recovery kinetics.
Fit to one exponential; time = 25 s; constraints: plateau < 1 and normalized fluorescence set to 0 at time zero.
Fit to two exponentials; time = 120 s; constraints: plateau < 1 and normalized fluorescence set to 0 at time zero.
Fit to two exponentials; time = 90 s; constraints: plateau < 1 and normalized fluorescence set to 0 at time zero.
FRAP t1/2 for fast and slow components of the recovery curves. SDs were calculated from the 95% CIs. Percentages corresponding to fast and slow recoveries are shown in parentheses.