Co-localization analysis between NES ^(2x) -mNG ^HO -(2x)DdSNXA ^PX and the different endosomal markers in HEK293-AT1 cells. Cells were transfected with NES^(2x)-mNG^HO-(2x)DdSNXA^PX and the mCherry-tagged indicated endosomal marker or LysoTracker Red, and z-stacks of confocal images were recorded from individual cells. A mask was then generated using the thresholded PI(3,5)P₂ image and used to calculate the average pixel intensities of the background-subtracted endosomal marker image in that mask throughout the z-planes. These values were then divided by the respective average pixel intensity values obtained for the entire thresholded endosomal marker throughout the z-planes. (A and B) Fractional co-localization values for the individual cells across the individual z-planes as box-and-whisker diagrams, with (B) average values analyzed using Welch’s ANOVA test with multiple comparison.