FRET for measurement of localized cAMP was performed as previously described (26 (link)). Briefly, coverslips with confluent L2 cells transiently transfected with pmEpac2 or cytEpac2 were placed in 300μl of oxygenated Locke’s medium (154mM NaCl, 5.6mM KCl, 2.2mM CaCl2, 1mM MgCl2, 6mM NaHCO3, 10mM glucose, 2mM HEPES) containing 0.05% BSA in a temperature-controlled (37 °C), modified Sykes-Moore Chamber mounted on a Nikon TE2000 inverted fluorescence microscope (Nikon Instruments, Inc., Melville, NY, USA). Cells were imaged under a 100× epifluorescence objective using a Xenon light source. Images were captured using NIS Elements 5.0 Imaging Software (Nikon Instruments, Inc., Melville, NY, USA). Cyan fluorescent protein (donor) fluorescence was measured using an excitation filter with 430–455 nm bandpass and an emission filter with of 470–490 bandpass. Yellow fluorescent protein (acceptor) fluorescence was measured using an excitation filter with 500–520 nm bandpass and an emission filter with of 535–565 nm bandpass. FRET was measured using an excitation filter with 430–455 nm bandpass (donor excitation) and an emission filter with 535–565 nm bandpass (acceptor emission). Average FRET intensity was estimated from corrected images. Changes in FRET are calculated as a percentage of basal FRET (%ΔF).