Fig. 5: Detection of ternary protein interactions.
a Model for the complementation of tripartite split-FAST and equations relating effective fluorogen concentration for half maximal complementation in the absence of interaction (EC50,–interaction) and in the presence of interaction (EC50,+interaction) to the efficacy of the self-complementation and of the interaction-dependent complementation. b, c Normalized average fluorescence of about 20,000 HEK293T cells co-expressing the triplet pFAST99-114–NFRB, CFRB–pFAST1-98 and FKBP–pFAST115-125 treated without or with 500 nM of rapamycin, and with 0, 1, 5, 10, 25 or 50 μM of HBR–2,5DM. Data represent the mean ± SD of two independent experiments. d–f HEK293T cells expressing the triplet pFAST99–114–NFRB, CFRB–pFAST1-98 and FKBP–pFAST115-125 were treated with 10 μM HBR-2,5DM. Cells were imaged after addition of 500 nM of rapamycin. Experiments were repeated three times with similar results. d Representative micrographs before and after addition of rapamycin (see also Supplementary Movie 3). Scale bars 20 μm. e Temporal evolution of the fluorescence signal (F/Fmax) after addition of rapamycin n = 45 cells from three independent experiments. f Fluorescence fold increase (Fmax/Fmin) upon addition of rapamycin. g–i HEK293T cells expressing the triplet pFAST99–114–NFRB–ECFP–CAAX, Lyn11-mCherry–CFRB–pFAST1–98 and FKBP–pFAST115–125 were treated with 10 μM HBR-2,5DM. Cells were imaged after addition of 500 nM of rapamycin. Experiments were repeated three times with similar results. h Representative micrographs before and after addition of rapamycin (see also Supplementary Movie 4). Scale bars 20 μm. i Temporal evolution of the fluorescence signal (F/Fmax) after addition of rapamycin n = 20 cells from three independent experiments. e, f, i Each cell is color-coded according to the biological replicate it came from. e, i The dot lines represent the mean value of each biological replicate, while the black line represents the mean of the three biological replicates. f The solid circles correspond to the mean of each biological replicate. The black line represents the mean ± SD of the three biological replicates. Source data are provided as a Source Data file.
