PI(3)P and its lipid kinase VPS34 colocalize with SNX17 and SNX27 in dendrites. (A) Domain architecture of the SNX-FERM proteins used in this study. (B) Rat hippocampal neurons were transfected at DIV16 with dsRed-EEA1-FYVE and either GFP-SNX17 or GFP-SNX27 and fixed 24 h later. Scale bar, 5 µm. (C) The colocalization between dsRed-EEA1-FYVE– and either GFP-SNX17– or GFP-SNX27–positive puncta was analyzed using Mander’s colocalization coefficient (×100). GFP-SNX17: 79.55 ± 1.617%, N = 30 neurons; GFP-SNX27: 83.00 ± 1.573%, N = 30 neurons. (D) Rat hippocampal neurons were co-transfected at DIV16 with mScarlet-SNX27, mNeonGreen-SNX17, and HA-VPS34. 24 h later, cells were fixed, permeabilized, and immunostained with an anti-HA antibody. Scale bar, 2.5 µm. (E) The percentage of mNeonGreen-SNX17 overlapping with mScarlet-SNX27 was analyzed using Mander’s colocalization coefficient (×100). 81.22 ± 3.386%, N = 15 neurons. Three independent experiments. Error bar is SEM. (F) The percentage of mScarlet-SNX27 overlapping with mNeonGreen-SNX17 was analyzed using Mander’s colocalization coefficient (×100). 82.49 ± 2.480%, N = 15 neurons. Three independent experiments. Error bar is SEM. (G) The percentage of endosomes containing both mNeonGreen-SNX17 and mScarlet-SNX27 that colocalize with HA-VPS34 was analyzed using Mander’s colocalization coefficient (×100). 72.83 ± 3.323%, N = 15 neurons. Three independent experiments. Error bar is SEM. DIV, days in vitro.