Analysis of Sam68-binding SH3 domains in vivo by FRET-analysis.
Expression constructs for CFP-tagged Sam68 and YFP-tagged SH3 domains were used to co-transfect 293T cells as indicated. 48 h post transfection, cells were harvested for flow cytometric analysis. Direct protein interaction in vivo was assayed by determining FRET from CFP to YFP by exciting CFP at 405 nm and measuring fluorescence with filters 450/50 (CFP only) vs. 585/42 (CFP + YFP-FRET-signal). (A) Representative diagrams showing the shift of cell populations as a result of FRET. Based on the negative control (CFP, or CFP-Sam68, and YFP on separate plasmids) and the positive control (CFP-YFP-fusion protein on one plasmid) two gates were defined, enclosing cells that do not exhibit FRET (R2, red), or that do exhibit FRET (R3, green), which is manifest by a shift to the left (i.e. lower CFP emission) and simultaneously to the top (i.e. higher YFP-emission). The degree of this shift depends on the FRET-efficiency. (B) FRET signals for all domains assayed. Results are shown as mean ± standard deviation from three independent experiments.