Detection of PI(3,5)P ₂ using the NES ^(2x) -mNG ^HO -(2x)DdSNXA ^PX construct in HEK293-AT1 cells. (A–D) Confocal images of HEK293-AT1 cells transfected with the high-avidity PI(3,5)P₂ probe, NES-mNG^HO-(2x)DdSNXA^PX, and mCherry-tagged Rab5 (A), mCherry-Rab7 (B), and LAMP1-mCherry (C), or co-stained with LysoTracker Red (D). Importantly, in these cells, PI(3,5)P₂-positive endosomes were apparent even without expressing the PIKfyve^CC fragment. Note that the brightest PI(3,5)P₂ signal is not associated with Rab5-positive endosomes and shows the closest co-localization with only a fraction of the Rab7 endosomes. While the PI(3,5)P₂ signal is also associated with a fraction of LAMP1-positive endosomes, the bright “speckles” emanating from the endosome proper only show overlap with Rab7 but not LAMP1. Also, note that the majority of the PI(3,5)P₂ signal does not co-localize with the signal from the LysoTracker Red; however, in some cells the green signal can be seen within the enlarged lysosomes (white arrows). For quantification of images similar to those shown in A–C, see Fig. S4 (scale bars = 5 µm).