FRET Visualization of Protein Interactions

FRET was visualized on an epifluorescence Carl Zeiss TM210 microscope using the three-filter method as described previously (24 (link)). The plasmid for YFP-HSP70 was generously provided by Harm Kampinga (University of Groningen, Netherlands). Cells were seeded on 29-mm glass bottom dishes and transfected with plasmids encoding YFP-HSP70 (0.2 μg) and wild type or mutant versions of CFP-SERT (0.8 μg). For experiments involving the HSP70 inhibitor VER-155008 (Sigma), the cells were incubated with the drug (40 μm) for 2 h prior to measuring FRET. The images were acquired using a 6× oil immersion objective and an automated filter wheel (Ludl Electronic Products, Hawthorne, NY) to allow for rapid switching between the fluorescence excitation and emission filters for CFP (I_CFP; excitation, 436 nm; emission, 480 nm; dichroic mirror, 455 nm), YFP (I_YFP; excitation, 500 nm; emission, 535 nm; dichroic mirror, 515 nm), and FRET (I_FRET; excitation, 436 nm; emission, 535 nm; dichroic mirror, 455 nm). The images were captured by a charge-coupled device camera and analyzed using the ImageJ PixFRET plug-in (25 (link)).