Assessment of the EGFP-tagged (2x)ML1N as a PI(3,5)P ₂ reporter. (A) Expression of the EGFP-tagged (2x)ML1N reporter (Li et al., 2013) in HEK293A cells shows localization of this probe to endocytic structures, which are not eliminated even by prolonged treatment with apilimod (1 µM). (B) Rab5-specific BRET-based biosensor that contains the (2x)ML1N segment (sLuc-(2x)ML1N-tPT2A-mVenus-Rab5) shows a weak but clear response to acute PI(3,5)P₂ generation by FKBP-PIKfyve^CC recruitment. Grand averages (±SEM) are presented from three experiments, with distinct treatments performed in triplicate within each replicate. To allow for direct comparisons, we included the response of the (2x)DdSNXA^PX BRET reporter shown in Fig. 8 D (gray symbols).