Figure 3. Characterization of mlCNBD-FRET in HEK293 cells.

(A). Representative Western blot using total protein lysates from mlCNBD-FRET and mlCNBD-FRET-R307Q-expressing cells, stained with an anti-His and an anti-GFP antibody. Calnexin (Cal) has been used as a loading control. (B) Immunocytochemistry. HEK293 cells expressing mlCNBD-FRET (cerulean: blue, citrine: green) have been labeled with an anti-GFP antibody and a fluorescent secondary antibody (red). Scale bar 25 µm. (C) Ligand binding of cAMP to mlCNBD-FRET in HEK293 cells determined by fluorescence spectroscopy. Representative experiment showing an increase of the baseline-corrected cerulean/citrine emission ratio ΔF of mlCNBD-FRET (430 nm excitation) at different cAMP concentrations. Cells have been permeabilized with 20 μM digitonin before addition of cAMP. Data have been fitted using a single binding-site model (red line) (Cukkemane et al., 2007). (D) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with 40 μM NKH477/500 μM IBMX. FRET has been measured by fluorescence microscopy. Representative traces of raw data are shown above. HEK293 cells expressing mlCNBD-FRET-R307Q (grey) have been used as a control. Data are presented as mean ± S.D. (mlCNBD-FRET: n = 31; mlCNBD-FRETR307Q: n = 3). (E) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with 2 μM isoproterenol. Representative traces for the raw data are shown above. Data are presented as mean ± S.D.; n = 9. (F) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with 3 mM SNP. Representative traces for the raw data are shown above. Data are presented as mean ± S.D.; n = 36. (G) Changes in FRET in HEK293 cells expressing mlCNBD-FRET (black) or mlCNBD-FRET-R307Q (grey) after stimulation with 40 μM NKH477. After reaching a steady-state, cells have been permeabilized using 1 μM digitonin. FRET has been measured using spectrofluorometer. Data are presented as mean ± S.D.; n = 3. (H) Changes in cerulean fluorescence lifetime using FLIM. HEK293 cells expressing mlCNBD-FRET were imaged under basal conditions, after addition of 20 μM digitonin, and the following addition of 5 μM cAMP. The cerulean fluorescence decay was recorded and fitted with a bi-exponential decay to calculate the lifetime. Data are presented as mean ± S.D. Representative images are shown using a look-up table ranging from 2.1 ns (blue) to 2.7 ns (red). (I) Mean values of the two lifetimes averaged over different regions of interest in part (H); n = 8 for each condition.

DOI: http://dx.doi.org/10.7554/eLife.14052.005

Figure 3—figure supplement 1. Characterisation of mlCNBD-FRET in HEK293 cells.

(A) Calibration of mlCNBD-FRET in HEK293 cells. FRET was measured in a cuvette in a spectrofluorometer under basal conditions (ES), after permeabilization with 20 μM digitonin, and the following addition of increasing cAMP concentrations (in nM). According to the null-point calibration method, the difference in FRET ratio of the treated samples to the basal condition was determined and used to determine the basal cAMP concentration. (B) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with 40 μM NKH477/500 μM IBMX (black). FRET has been measured using spectrofluorometer. Data are presented as mean ± S.D. DMSO (0.13%, red) has been used as a control; n = 3 for each condition. (C) Similar to part (B) after stimulation with 2 μM isoproterenol (black). (D) Similar to part (B) for stimulation with 3 mM SNP (black). (E) Change in the cerulean fluorescence lifetime measured using Fluorescence Lifetime Spectroscopy (FLS). Cells have been permeabilized with 20 μM digitonin followed by addition of cAMP. The cerulean fluorescence decay was recorded and fitted with a bi-exponential decay to calculate the lifetime. The mean of the two lifetimes was calculated and averaged over n = 3 experiments. (F) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after stimulation with different concentrations of NKH477. The FRET ratio has been calculated when reaching a maximum and normalized to the baseline ratio. Data is shown as mean ± S.D.; n = 4. (G) Changes in FRET in HEK293 cells expressing mlCNBD-FRET after alternatingly stimulating with 500 nM isoproterenol (black) followed by a wash-out with ES. As a control, cells were stimulated with buffer only (red). Data is shown as mean ± S.D.; n = 3.

DOI: http://dx.doi.org/10.7554/eLife.14052.006