Supplementary Figure 6: Quantification of the ERK activity (FRET/CFP) in HeLa cells expressing 2paRAF variants with P2A and full-length RAF1, IRES and full-length RAF1, or P2A and the RAF1 catalytic domain (a.a. 307-648) before and after the 2P activation. | Nature Methods

Supplementary Figure 6: Quantification of the ERK activity (FRET/CFP) in HeLa cells expressing 2paRAF variants with P2A and full-length RAF1, IRES and full-length RAF1, or P2A and the RAF1 catalytic domain (a.a. 307-648) before and after the 2P activation.

From: FRET-assisted photoactivation of flavoproteins for in vivo two-photon optogenetics

Supplementary Figure 6

HeLa cells stably expressing EKAREV-nls, an ERK FRET biosensor, were transfected with the plasmids encoding 2paRAF connected with full-length RAF1 or the RAF1 catalytic domain (a.a. 307–648) via P2A or IRES. Cells were photoactivated and imaged by 2P excitation (840 nm, 10mW, 0.828 μm/pixel, 2 µs/pixel, 20 scans). The ERK activity was plotted against mScarlet-I intensity to examine the correlation between the expression level of the plasmid and ERK activity. The ERK activity (FRET/CFP) in each cell before (black dots) and after (magenta dots) 2P activation (840 nm, 10 mW, 0.828 μm/pixel, 2 µs/pixel, 20 scans) was quantified and plotted as a function of mScarlet-I intensity; n = 21 cells; Representative data among n = 2 independent experiments. a.u., arbitrary units.

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