Fig. 3

FLIM-FRET analysis of proteins interacting with YULINK. A Plasmid construct for full-length YULINK protein containing four conserved WD40 repeats (blue) and 3 potential WD40 candidates (purple). Other plasmid constructs for truncated forms of N- or C-termini (YULINK∆N or YULINK∆C) are also shown. B–D To validate these interactions, YULINK (full-length or truncated forms) was conjugated with AcGFP and the interacting protein candidates, EPS15, RAB33B, or TICAM2, were conjugated with DsRed, separately. Then, they were co-expressed in HEK-293 T cells and the FRET (Förster Resonance Energy Transfer) of AcGFP was measured using multi-photon fluorescence lifetime imaging microscopy (FLIM). The mean fluorescence lifetime (τ) and the FRET efficiency E (%) were measured at 48 h after co-transfection of HEK-293 T cells with AcGFP-YULINK (full-length or truncated forms) and DsRed-EPS15, DsRed-RAB33B, or DsRed-TICAM2, respectively. The less mean fluorescence lifetime (τ) and more FRET efficiency E (%) indicated stronger interaction and shorter distance between AcGFP and DsRed. The fluorescence lifetime (τ) of cells expressing only AcGFP-YULINK was as a FLIM-FRET control