Fig. 4. Quantitative iFLAP. (A) HepG2 cells co-transfected with STAT3-CY and v-Src were analyzed in a perfusion chamber for live cell imaging at the confocal microscope. CFP and YFP fluorescence intensities were monitored exclusively in regions of interest (ROI, numbered red circles in the images) over time. The initial cytoplasmic bleaching was performed in the bleach ROI (white circle) over the time indicated by the red bar on the x-axis in the diagram. The diagram represents the mean CFP (blue lines) and YFP fluorescence intensities (orange lines) in the three ROIs over time. The red numbers in the diagrams correspond to the cytoplasmic (1), nuclear (2) and background (3) detection ROIs. A representative experiment is shown. (B) iFLAP experiments were performed with HepG2 cells transiently transfected with STAT3-CY and with stably transfected MEF^/-STAT3-CY cells co-transfected with v-Src or empty vector as described in A. The resulting curves were analyzed by mathematical modelling and computational parameter estimation. The bar chart depicts the mean values and s.d. of the exponential coefficient as a measure for the rate of shuttling.