Fig. 1
The FlAsH-based FRET DVL3 sensors. a The general scheme of the FlAsH-based FRET in vivo approach (in further detail in Supplementary Fig. 1a). b The design of four DVL3 FlAsH I–IV sensors with the CCPGCC tag and N-terminal ECFP tag. The insertions of the CCPGCC tag were placed at the positions with highest disordered prediction scores (PONDR-Fit21)—one CCPGCC tag per a linker region (FlAsH I, II, III) and the C-terminus (FlAsH IV). Multiple sequence alignment of the Dvl/DVL sequences at the site of insertion is shown below. Residues with >80% similarity are highlighted. Sequence of human DVL3 used for cloning as a template is shown in red box. c Biological properties of four ECFP-DVL3 FlAsH sensors are indistinguishable from wild-type ECFP-DVL3 (for details see Supplementary Fig. 1c–e). d The intramolecular and intermolecular FRET efficiency in the DVL3 FlAsH I–IV sensors in HEK293 wild-type cells. The position of the fluorophores in DVL3 molecules in both experimental setups are schematized above the graph. One data point corresponds to one analyzed cell. Data from three independent transfections were merged. Data in d represent median ± interquartile range. FRET eff., FRET efficiency; BAL, British anti-Lewisite; ECFP enhanced cyan fluorescent protein
