Fluorescent Protein Co-Localization Assay

The DNA transfection followed a Promega FuGENE protocol by the manufacturer as described in Refs. 45 (link), 59 (link), and 60 (link). The HEK293 cells were transfected after reaching 30–50% confluence in a LabTeck 4-well cover glass chamber with the mixtures of bGCAP-GFP or hRD3-GFP coding plasmids with mOrange-RetGC1 coding plasmid, at ∼1/100 molar ratio (58 (link), 59 (link), 60 (link)), using 3 μl of Promega FuGENE reagent per 1 μg of DNA. Confocal images were taken after 24–30 h of incubation in 5% CO₂ at 37 °C, using an Olympus FV1000 Spectral instrument with the respective 543- and 488-nm excitation for the red and the green fluorochromes in sequential mode. The images were processed, and the PCC values in whole-cell images were determined using Olympus FluoView FV10-ASW software as previously described (43 (link), 44 (link), 45 (link), 58 (link), 59 (link), 60 (link)). No changes to the original images were made except for minor γ correction applied to whole image for more clear presentation in print. Quantitative analyses were performed using the original images without corrections.