Table I.
Normalized FRETN c for CFP donor and YFP acceptor pairs used to characterize AKAP79, CaNA, and PKA-RII binding interactions in live cells
| CFP donor | YFP acceptor | |||
|---|---|---|---|---|
| AKAP79–YFP | CaNA–YFP | PKA-RII–YFP | AKAP18(1–16)–YFP | |
| AKAP79–CFP | <0 | 2.8 ± 0.8 | 12.1 ± 1.1 | <0 |
| AKAP79ΔCaN–CFP | − | <0 | − | − |
| AKAP79ΔPKA–CFP | − | − | <0 | − |
| CaNA–CFP | 1.4 ± 0.4 | − | − | − |
| PKA-RII–CFP | 13.8 ± 3.0 | − | − | − |
| AKAP79WT + CaNA–CFP | − | − | 9.1 ± 1.6 | − |
| AKAP79ΔCaN + CaNA–CFP | − | − | <0 | − |
| AKAP79ΔPKA + CaN–CFP | − | − | <0 | − |
| AKAP79ΔCaN + ΔPKA + CaN–CFP | − | − | <0 | − |
| AKAP79(321–360)–CFP | − | 13.3 ± 1.0 | − | − |
| AKAP18(1–16)AKAP79(321–360)–CFP | − | 35.6 ± 10.0 | − | − |
| AKAP18(1–16)–CFP | − | <0 | − | − |
Mean fluorescence intensities in arbitrary linear units were measured (see Materials and methods) for CFP, YFP, and raw, uncorrected CYFRET for regions of CFP and YFP overlap in membrane ruffles and vesicles. For each image, corrected FRETc was calculated from raw CYFRET by fractional subtraction (see Materials and methods) of CFP and YFP intensity values. The FRETc intensity values were then normalized (FRETNc) to the amounts of CFP donor and YFP acceptor present by the product (CFP)(YFP). FRETNc is thus proportional to Keq = [donorCFP · acceptorYFP]/[donorCFP][acceptorYFP], the equilibrium constant for the binding interaction. FRETNc values are represented above as the mean ± SEM (×10−5) for multiple images (n = 5–8) for each donor–acceptor pairing. Negative FRETc values are seen for CFP–YFP pairs that fail to exhibit FRET due to bleedthrough overcorrection (see Materials and methods) that prevents false positive FRET measurements. Negative FRET pairs are thus represented by FRETNc values < 0 above. Thus, positive FRETNc values in this table and the FRETc images shown in the figures are actually slight underestimates of the actual FRET signals.