Figure 4. Chemical induction of synthetic ERK signal oscillation. (a) Schematic illustration of 2 the experimental setup. The washing procedure was carried out using a culture-medium flow 3 system as shown in Figure S13a. (b) Representative time-lapse confocal fluorescence images of 4 a HeLa cell coexpressing cRaf-mNG-iK6DHFR and mCh-ERK. Images were taken at the time 5 points indicated by the asterisks shown in panel c. mDcTMP and TMP were added at 6 concentrations of 10 and 50 µM, respectively. During the washing step (the blue bar in panel c), 7 fresh medium continuously flowed at a rate of 1 mL/min. Scale bar, 20 μm. For the time-lapse 8 movie, see Movie S8. (c) Time course of cRaf-mNG-iK6DHFR translocation and ERK activity. To 9 evaluate cRaf-mNG-iK6DHFR translocation (top), the normalized fluorescence intensity of cRaf-10 mNG-iK6DHFR in the cytoplasm was plotted as a function of time. To evaluate ERK activity 11 (bottom), the normalized ratios of the nuclear fluorescence intensity to the cytoplasmic 12 fluorescence intensity (N/C ratios) of mCh-ERK were plotted as a function of time. Data are 13 presented as the mean ± SD (n = 12 cells). 14 15
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