TABLE 1.
Cdc42 FRET analysis The table shows FRET efficiency obtained for Cdc42V12/N17-effector interactions by AP-FRET (first column) and changes in lifetime obtained with FLIM (third column). Data in the second column represent cross-correlation values for changes in mRFP/GFP intensity monitored during bleaching in AP-FRET. Data are expressed as averages ± S.D. (n = 7–12).
| Construct | AP-FRET efficiency ± S.D. | Cross-correlation value ± S.D.a | FLIM-FRET efficiency ± S.D. |
|---|---|---|---|
| % | % | ||
| Positive control | |||
| mRFP-GFP (tandem fusion) | 28.6 ± 3.7 | –0.99 ± 0.01 | 25.4 ± 7.4 |
| Negative controls | |||
| Cyto-mRFP/GFP | 1.9 ± 1.5 | 0.17 ± 0.63 | 1.1 ± 1.2 |
| mRFP-IRSp53 + cyto-GFP | 2.1 ± 1.5 | –0.16 ± 0.55 | |
| GFP-N-WASP + cyto-mRFP | 2.7 ± 1.9 | –0.63 ± 0.42 | |
| Experimental | |||
| mRFP-Cdc42V12 + GFP-CRIB (domain) | 18.4 ± 3.6 | –0.99 ± 0.01 | 22.7 ± 5.5 |
| mRFP-Cdc42N17 + GFP-CRIB (domain) | 2.3 ± 2.3 | –0.09 ± 0.75 | 4.1 ± 1.8 |
| mRFP-N-WASP + GFP-Cdc42V12 | 10.2 ± 2.4 | –0.97 ± 0.02 | 11.7 ± 2.4 |
| mRFP-N-WASP + GFP-Cdc42N17 | 2.4 ± 1.7 | –0.47 ± 0.49 | 6.0 ± 2.2 |
| mRFP-IRSp53 + GFP-Cdc42V12 | 9.8 ± 3.5 | –0.94 ± 0.06 | 13.8 ± 4.2 |
| mRFP-IRSp53 + GFP-Cdc42N17 | 2.7 ± 2.7 | –0.10 ± 0.69 | 3.5 ± 2.6 |
a
n = 7–12 cells for each pair. Cross-correlation values between –0.7 and –1.0 are considered as positive FRET (12)