Supplementary Figure 3: Live-cell fluorescence resonance energy transfer (FRET) analysis of interactions between YFP-histones and CFP-BRD proteins.
From: BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones
a, Diagram of CFP-BRD proteins. b, Three-dimensional (3D) surface plots of FRET signals from HEK293T cells transfected with YFP-H4 alone (i) or CFP-BRD4short alone (ii). In flow cytometric measurements, spillover signals (e.g., false FRET signals due to spillover of CFP signals into the FRET detector channel) were digitally compensated so that YFP- or CFP-protein alone exhibited only marginal FRET signals. c, 3D surface plots displaying FRET signals covering wide ranges in CFP/YFP protein expression. HEK293T cells were transfected with YFP-H4 and CFP-BRD proteins as indicated. The same spillover-compensation matrix set in b was applied to all plots. d, Cells were transfected with expression plasmids for CFP-BRD proteins and YFP-histones or an empty vector in combinations as indicated. Data were collected using a fixed window set for similar levels of YFP-proteins, and FRET signals (means ± SEM) were plotted against CFP signals. For cells transfected with CFP-BRD plasmids and an empty vector, all data were collected.
