Figure 4

FRET evaluation of ECF-HIF-1α and PHD1-EYFP interaction. Embryonic kidney cancer cells HEK 293 were transfected with plasmids expressing the donor molecule ECFP-HIF-1α and the acceptor PHD1-EYFP. 48 h later cells were scanned for fluorescence and FRET was determined. The black curve indicates random FRET efficiency values as described in detail in Figure 3. Filled circles show FRET values from individual cells which result in mean FRET efficiency for HIF-1α/PHD1 as indicated by the red and blue curves. Values for the red curve were obtained after treatment of the cells with the PHD1 inhibitor dimethyloxalylglycine (0.5 mM for 24 h) while the blue curve shows values from untreated controls. The inserts show donor (ECFP-HIF-1α) and acceptor (PHD1-EYFP) fluorescence and the resulting FRET efficiency exclusively located in the nuclei of the cells.