Figure 2. Chemogenetic activation of the Raf/ERK pathway. (a) Schematic illustration of the 2 experimental setup. (b) Confocal fluorescence images of HeLa cells coexpressing EGFP-3 iK6DHFR-cRaf and ERK-KTR-mKO were taken before (left) and 60 min after the addition of 4 mDcTMP (10 µM) (right). Scale bar, 20 μm. For the time-lapse movie, see Movie S2. (c) Time 5 course of EGFP- iK6DHFR-cRaf translocation and ERK activation. To evaluate EGFP- iK6DHFR-6 cRaf translocation (bottom), the normalized fluorescence intensity of EGFP-iK6DHFR-cRaf in the 7 cytoplasm was plotted as a function of time. To evaluate ERK activity (top), the normalized ratios 8 of the cytoplasmic fluorescence intensity to the nuclear fluorescence intensity (C/N ratios) of 9 ERK-KTR-mKO were plotted as a function of time. Data from control experiments using EGFP-10 iK6DHFR-cRaf in the presence of the MEK inhibitor PD184352 (Figure S7a) or EGFP-iK6DHFR 11 (lacking the cRaf protein) (Figure S7b) were plotted on the same graph. Data are presented as 12 the mean ± SD (n = 5 cells). 13 14
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