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To test the proteins for photoreactivity, we first measured the "dark" absorbance of the protein, e.g., the naive protein that has been incubated and regenerated with the chromophore (11-cis retinal or all-trans retinal).
The maximum absorbance of the 2 retinals is very similar when the apoprotein is not present: 380 nm for all-trans retinal and 379 nm for 11-cis retinal in ethanol.
To verify whether the two GPCRs were indeed able to form photopigments in a manner similar to opsin proteins, the two proteins were expressed in HEK293T cells and incubated with either 11-cis retinal or all-trans retinal. Previous work has shown that opsins with a long C-terminus express more efficiently in vitro if part of the C-terminus is removed (Terakita et al., 2008; Koyanagi et al., 2013).
Larvae expressing Channel-rhodopsin-2 (ChR2) in class IV neurons were fed with all-trans retinal, which is essential for the synthesis of ChR2 (Nagel et al, 2003).
Control animals, which were not fed all-trans retinal, did not display a reproducible avoidance of 488-nm light, but rather a stochastic distribution on both sides of the plate (Fig.