Fig. 4. N-WASP activity of PC12 cells imaged using a FRET biosensor: Exo70 antagonises NGF-induced N-WASP activation. Fluorescence intensities of CFP (Donor) and YFP (Acceptor) fluorophores of the CFP-N-WASP-YFP FRET biosensor upon excitation of the CFP moiety and CFP:YFP emission ratios (FRET ratio) are shown for representative cells of the different conditions. Cumulative FRET ratio histograms are shown for each condition. The FRET ratio is represented in false colour from 0.35 (blue) to 0.9 (red), indicating high to low FRET ratios, respectively. The blue trace represents the FRET ratio distribution in the absence of NGF treatment, the red trace that after NGF differentiation. The average ± s.e.m. of the distributions (and number of cells) are indicated in the respective colour. Statistical significance for the averages is indicated above the distributions. (A) Cells expressing the biosensor only, (B) cells co-expressing FLAG-tagged Exo70 and (D) cells transfected with siRNA for Exo70 knockdown. Bars, 5 µm (untreated cells), 10 µm (NGF-treated cells). (C) Irregularity indexes (average ± s.e.m.) of NGF-induced PC12 cells transfected with control or Exo70 siRNA (left). The western blot shows the efficiency of siRNA-mediated knockdown of Exo70 (70% as judged by densitometry) for Exo70 relative to actin (right).