Fig. 3: Evaluation of split site 98-99.

a Model for complementation of bipartite split-FAST and equations relating effective fluorogen concentration for half maximal complementation in the absence of interaction (EC_{50,–interaction}) and in the presence of interaction (EC₅₀,_+interaction) to the efficacy of the self-complementation and of the interaction-dependent complementation. b–e Normalized average fluorescence of about 20,000 HEK293T cells co-expressing the FK506-binding protein (FKBP) fused to the N-terminus of FAST_99–125 (b, c) or ^RspAFAST_99-125 (d, e) and the FKBP-rapamycin-binding domain of mammalian target of rapamycin (FRB) fused to the C-terminus of FAST_1–98 (b, c) or ^RspAFAST_1–98 (d, e) treated without or with 500 nM of rapamycin, and with 0, 1, 5, 10, 25 or 50 μM of HMBR. Data in c,e represent the mean ± SD of three independent experiments. f–h HEK293T cells expressing ^RspAFAST_1–98-FRB and FKBP-^RspAFAST_99-125 were treated with 10 μM HMBR. Cells were imaged after the addition of 500 nM of rapamycin. Experiments were repeated two times with similar results. f Representative micrographs before and after addition of rapamycin (see also Supplementary Movie 1). Scale bars 20 μm. g Temporal evolution of the fluorescence signal (F/F_max) after addition of rapamycin n = 33 cells from two independent experiments. F is the fluorescence intensity at time t, and F_max is the maximal fluorescence intensity reached during the time-lapse. h Fluorescence fold increase (F_max/F_min) upon addition of rapamycin. F_min is the fluorescence intensity at time t = 0, and F_max is the maximal fluorescence intensity reached during the time-lapse. g, h Each cell is color-coded according to the biological replicate it came from. g The dot lines represent the mean value of each biological replicate, while the black line represents the mean of the two biological replicates. h The solid circles correspond to the mean of each biological replicate. The black line represents the mean ± SD of the two biological replicates. Source data are provided as a Source Data file.