Detection of PI(3,5)P ₂ using DdSNXA ^PX domain variants. (A and B) Confocal images of HEK293A cells expressing the single (A) or tandem (B) version of the DdSNXA^PX probe fused to mNG^HO. In these cells, no endocytic structures were decorated with either construct regardless of how low the relative expression levels were. In most cells, a bright dot was visible at the centrosome region that did not disappear after apilimod treatment (1 µM). Similarly, the nuclear and nucleolar enrichment was also not affected by apilimod treatment. In subsequent versions of the probes, a tandem NES^(2x) was added to prevent the prominent nuclear localization. (C) Co-expression of the FKBP-PIKfyve^CC fragment with the tandem NES^(2x)-mNG^HO-(2x)DdSNXA^PX shows localization of the reporter to endocytic structures (scale bars = 10 µm).