Figure 1
A sensitive method for PPI interaction studies based on BRET saturation assays. (A,B) HEK-293T cells were transfected with 0.025 to 25 ng of YFP-Nluc plasmid per well. Nluc bioluminescence was quantified using various microplate readers (A), and YFP fluorescence was quantified using microplate readers or an automated confocal microscope (B). (C) YFP and Nluc signals were sequentially quantified for 13 sets of plasmids transfected in HEK-293T cells at 1:1 A:D ratios. (D) Bioluminescence spectra properties of reference proteins. Results are the average of three independent experiments. (E) Sequential measurement of the fluorescence of the acceptor (with an automated microscope) and the bioluminescence emitted from both A:D upon BRET (with a plate reader) in order to calculate the net BRET and the A:D expression ratio using the BRET-free Nluc-block-YFP control for BRET saturation assays. (F) Broad range transfection method for BRET saturation assay over 11 ratios (243:1 to 1:243, see Supplementary Fig. S2). (G) Diagram of NF-κB protein domains and sequence homologies. (H,I) BRET saturation assay in HEK-293T cells transfected with the donor (Nluc-p50) and acceptor (YFP-tagged) plasmids using fixed (H) or variable (I) donor. The net BRET was plotted with the YFP/Nluc expression ratios and fitted using a non-linear regression curve to determine both BRETmax and BRET50. The results represent four independent biological replicates, each involving three technical replicates. BRETmax values (J,K) were used to define interacting, non-interactive pairs (grey labeled) and extrapolate a 3 σ-threshold [(J) 0.055 and (K) 0.023]. BRET50 values were reported only for significant interacting pairs (L,M). # not displayed.
