Fig. 6. (A) Structure of the hit compound, 5c{3,9}. (B) ELISA-based evaluation of LRS and RagD stabilization by 5c{3,9} in a dose-dependent manner. (C) Schematic representation of FRET between LRS-CFP and RagD-YFP. When LRS directly interacts with RagD, LRS-fused CFP is physically close to RagD-fused YFP, generating a FRET signal. Fluorescence of 475 nm from CFP with excitation at 433 nm transfers to YFP and emits fluorescence at 527 nm. (D) Relative FRET efficiency ratio between LRS-CFP and RagD-YFP in HEK293T cells co-transfected with LRS-CFP and RagD-YFP. Images were taken at 10 min intervals over 3.5 h in a single cell. Cells were randomly selected from 3 independent experiments (P = 0.002; Mann–Whitney Test). 5c{3,9} (clear square) or 5a{2,9} (reverse black triangle) was treated at a 40 μM concentration after an initial 30 min of live cell imaging. Images were captured with CFP/CFP, CFP/YFP, and YFP/YFP filters (excitation/emission). Captured FRET images were analyzed using Softworks (imaging software) to exclude false-positive signal such as CFP crosstalk and YFP crosstalk. (E) Representative calculated FRET within cells. The color scale indicates the range of FRET intensity, from low (blue) to high (red). Scale bar, 20 μm.