FIG. 8.
Binding of GSH-monochlorobimane and DOX by FRET analysis. Binding of GSH-monochlorobimane (GSH-MCB) and DOX was studied in MEF−/− and MEF−/− transfected with RLIP76-GFP vector. Cells grown on the sterilized coverslips were treated with either 50 μmol/l monochlorobimane or 10 μmol/l DOX and incubated at 37°C for 20 min. Cells were fixed with 4% paraformaldehyde, and FRET and molecular interactions in the cell were analyzed using time-resolved confocal microscope MT 200 (Picoquant) with pulsed diode laser excitations at 405 nm (for MCB donor) and 475 nm (for GFP donor). For MCB observation, 465-nm (10-nm bandwidth) interference filter crossed with 430-nm long path cutoff was used and for GFP observation, 490- to 530-nm interference filter crossed with 500-nm long path filter was used. The top images show the observed intensities for respective samples (A). Below the histogram, the graph presents observed fluorescence lifetime measured for the respective images. For MCB alone (histogram in red), the signal is very bright and fluorescence lifetime is long, as expected, ∼9 ns with the relatively narrow distribution. Addition of RLIP76-GFP results in dramatic shortening of MCB fluorescence lifetime, which dropped to ∼4 ns (histogram in red) (B). The validity of the FRET analysis was confirmed using the fluorescent anthracycline drug, DOX, which is known to bind to RLIP76. C: Lifetime histogram for GFP only (green) and fluorescence of GFP in the presence of DOX (blue). The fluorescence lifetime of GFP in the presence of DOX is shorter, confirming that DOX clearly did interact with the green fluorescence protein of RLIP76. (A high-quality digital representation of this figure is available in the online issue.)