FACS-FRET Measurements and Analysis

FACS-FRET measurements were performed using a FACSAria (BD Bioscience) equipped with 405 nm, 488 nm and 633 nm lasers. To measure ECFP and FRET cells were excited with the 405 nm laser and fluorescence was collected in the ECFP channel with a standard 450/40 filter, while the FRET-signal was measured with a 529/24 filter (Semrock). To measure EYFP, cells were excited with the 488 nm laser while emission was also taken with a 529/24 filter (Semrock). For each sample, we evaluated a minimum of one thousand CFP/YFP positive cells that fell within the background adjusted gate (Fig. 1a, panel 2). To analyse subcellular localization or FRET via confocal microscopy, transfected cells grown on coverslips were mounted on microscope slides using mowiol mounting solution (2.4 g polyvinylalcohol, 6 g Glycerin, 18 ml PBS) and imaged with a Zeiss LSM510 Meta. Confocal FRET analysis were performed as described in the “FRET and colocalization analyzer – Users guide”[7] (link).