Assembly of two proteins on a scaffold in live cells. (A) Schematic of multiprotein assembly. Plasmids encoding the indicated components were expressed in COS7 cells (Transfection). Self-assembly of the split GFP linker (step 1) recruits the SNAP-GFP11 component to the DmrA-scaffold-GFP(1–10), resulting in green fluorescence. Addition of A/C heterodimerizer (step 2) recruits the mCherry-DmrC component, resulting in FRET. (B and C) FRET donor (split GFP) and FRET acceptor (mCherry) components were recruited to scaffolds of 0 nm (GSG peptide), 5 nm SAH, or 10 nm SAH by the addition of A/C heterodimerizer for 1 h, and FRET was determined in live cells. (B) Representative calculated FRET efficiency (Ed) images. Yellow dotted lines indicate the outline of each cell. See Fig. S2 A for the associated raw images. Bar, 10 µm. (C) Calculated FRET efficiencies (Ed). n ≥ 31 cells in three independent experiments for each condition. ***, P < 0.0001; n.s., not significant as compared with the (−) A/C heterodimerizer condition. Data are presented as the average ± SEM (error bars).