Fig. 2

Colocalization analysis of CCR5 epitopes. A simplified example of how colocalization is quantified. When the NT or ECL2 CCR5 epitope antibody (red) labels a CCR5 C-terminus-GFP fusion protein (green), the overlapping fluorophores create a yellow signal. Unlabeled ectopically expressed CCR5-GFP fusion protein will show as green only and labeled endogenously expressed CCR5 will show as red only. The color identity and density spread are displayed in the graph along with the intensity thresholds set (white lines) using single color controls. The inset is a cartoon of which category a pixel within the image is assigned based on the thresholds, with 1 being green only, 2 being red only, 3 being yellow (overlap), and 4 being background (A). A cell (B) is analyzed using the ZEN Blue 2.3 software, and each of the colors are identified by the computer based on the parameters set. The extracellular CCR5 epitope in this example (NT; CTC8) is highlighted in orange, CCR5-GFP in cyan, and NT + C-terminus-GFP overlap in dark blue by the analysis software (C). The pixels in these identified regions are counted by the program, and the colocalization coefficient is calculated (dark blue/dark blue + cyan) to determine what percentage of CCR5-GFP was bound by the antibody. Bar size—5 µm