FRET efficiency between CaNA–CaM or CaN-CaNB represents CaN activation in HEK cells. A, FRET strategy that was used for detection of CaN–CaM interaction. B, Images of HEK cells transiently transfected with GFP-CaM and RFP-CaNA for 24 h. Cells were excited at GFP-specific excitation wavelength (488 nm). The FRET ratios were calculated by reporting emission intensity of RFP, measured at 565 nm, to the GFP emission peak measured at 522 nm. The pseudocolored ratio images (FRET images) represent the emission changes before (time, 0 min) and 30 min after the addition of 2 μm ionomycin. Cells treated with ionomycin for 30 min showed higher FRET compared with 0 min. The color scale (bottom right) indicates that a shift toward the red end of the spectrum corresponds to higher FRET efficiency. C, Quantitative analysis of normalized FRET ratio in HEK cells transiently transfected with GFP-CaM and RFP-CaNA for 24 h and treated with 2 μm ionomycin for 30 min. No change is detected when the donor alone (GFP-CaM) was transfected. D, To confirm the existence of a FRET signal between GFP-CaM and RFP-CaN, a photobleach experiment was performed. HEK cells were transfected with both GFP-CaM and RFP-CaNA and treated for 30 min with ionomycin. After fixation, the RFP fluorescent signal of a small cytoplasmic region of interest was photobleached with 100% excitation light at 543 nm for 5 min (top panel, yellow boxes), and the intensity of the GFP signal was measured before and after bleaching. As a control, the GFP intensity was quantified in nonbleached areas as well (top panel, blue boxes). An increase of the GFP-CaM emission intensity was observed in areas where the RFP signal was photobleached, but no change was detected in control areas. E, Endogenous change in FRET signal was quantified by staining the cells for endogenous CaNA-Alexa 488 and endogenous CaM or CaNB-Cy3. The top panel represents pseudocolored ratio images (FRET images) observed in HEK cells treated with ionomycin or DMSO as a vehicle control for 30 min. The bottom panel shows quantitative analysis of normalized spectral ratio (n ≥ 60 cells; *p < 0.05; **p < 0.001). F, FLIM analysis of the interaction between CaNA and CaM in HEK cells treated with 2 μm ionomycin for 30 min. After treatment, cells were stained with endogenous CaNA-Alexa 488 and endogenous CaM-Cy3. The top panel, on the left, shows intensity images of Alexa 488 fluorescence reflecting the pattern of endogenous CaNA. The top panel, on the right, shows pseudocolored FLIM images. The bottom panel shows quantitative FLIM analysis of in HEK cells treated with DMSO and ionomycin. Cells stained with CaNA-Alexa 488 were used as a negative control to establish Alexa 488 lifetime in the absence of an acceptor fluorophore (Cy3) (data not shown). Lifetime is in picoseconds; *p < 0.05; n ≥ 25 cells. Data represent mean ± SEM.