Figure 6.

Dependence of the cellular average of the apparent FRET efficiency (E_app) on the level of expression [log (F^D/a.u.)]. The cytoplasmic constructs ADAA (open circles), ADNN (solid circles), NDAN (open triangles), and NDNA (solid triangles) were expressed in CHO cells for different periods of time after transfection, as described in the text. The level of expression was estimated from the spectral properties of the donor. (a) Each point represents the cellular average of E_app corrected for acceptor direct excitation at image pixel level (before averaging) for a single cell transfected with one of the four constructs as shown. To avoid unnecessary clutter of the plots, the standard deviations were not plotted. (Lines) Obtained by linear regression, serving as a guide to the eyes. (b) Each point represents the difference between the measured and predicted values of E_app for ADAA (i.e., $\Delta E_{\text{app}}=E_{\text{app}}^{\text{corrected}}-E_{\text{predicted}}$, where $E_{\text{app}}^{\text{corrected}}$ and E_predicted are from Eqs. 18–22, respectively), plotted against the corresponding mean value of log (F^D/a.u.) for the range shown on the abscissa. (Vertical bars) Propagated errors computed by adding the individual errors in quadrature. To see this figure in color, go online.