PILS-Nir1 is a high-affinity PA biosensor that can be used to study endogenous PA signaling in a variety of contexts. (A–E) Comparison of PILS-Nir1 and NES-PABDx2-Spo20 responses at the PM after stimulation by 100 nM PMA (A), or at the mitochondria (B), Golgi (C), Rab5 endosomes (D), or ER (E) after recruitment of FKBP-PI-PLC and FKBP-DGKα with 1 μM rapamycin. Organelle markers are shown in gray. Graphs show the grand means ± SEM of 3–4 experiments with 30–54 cells. Data in A are replicated from Fig. 1, D and F. Data in B are replicated from Fig. 4 F. (F) Cos7 cells transfected with PILS-Nir1 and NES-PABDx2-Spo20 showed the biosensors’ response to 100 μM ATP. (G) HeLa cells were transfected with the biosensors to compare the response to treatment with 100 μM histamine. Graphs show grand means ± SEM for three to four experiments. A total of 33–57 cells were analyzed. (H) Nir2 MCS formation was quantified as the change in fluorescence at a given time (Ft) divided by the fluorescence before 100-μM CCh stimulation (Fpre). GFP-Nir2 was co-expressed with either iRFP-PILS-Nir1 or a control biosensor iRFP-Tubby(c), which binds PIP2. The graph shows the grand means ± SEM for 4–5 experimental replicates (n = 44–48 cells).