Fig. 5
From: FRET-based cyclic GMP biosensors measure low cGMP concentrations in cardiomyocytes and neurons
PfPKG biosensors display 100-fold selectivity of cGMP over cAMP. a, b Homogenate of HEK293 cells transfected with Yellow PfPKG biosensor (a) or Red PfPKG biosensor (b), incubated with IBMX (100 µM) and increasing concentrations of cAMP (black). Corresponding data for cGMP (gray) from Figs. 1c, 1e are included for direct comparison. FRET was measured as described in Materials and Methods and normalized to the minimum (absence of CN) and maximum (highest CN concentration). Data shown are representative of four independent experiments. c, d Recording of FRET ratio (CFP/Venus) in single HEK293 cells expressing the Yellow PfPKG biosensor and stimulated with isoprenaline (Iso; 10 µM) or the AC activator forskolin (Fsk; 25 μM) followed by SNAP (50 nM) and IBMX (100 µM). FRET signal was normalized to that prior to first stimulation. Data shown are representative of five (Iso-SNAP-IBMX) and five (Fsk-SNAP-IBMX) independent experiments. e, f Recording of FRET ratio in single cardiomyocytes expressing the indicated biosensor and stimulated with Iso (10 nM), followed by CNP (300 nM) and IBMX (100 µM). FRET signal was normalized to that prior to first stimulation. Data are representative of five cells from four animals (e) and six cells from four animals (f). g Quantification of data in e-f. Data are mean ± SEM. *p < 0.05 vs. “Iso” (two-way ANOVA with Tukey post hoc test)
