PTENA, PTEND, and the functional PI(3,5)P₂ binders fused to GFP are not enriched near the cell tip. (A) Localization of PTENA, PTEND, and the PI(3,5)P₂ binders is cytosolic, like GFP alone. The PTEN domain of For2A (second panel) is tip enriched. (B) Immunoblot using an anti-GFP antibody shows that the full-length fusion proteins are expressed. Numbers are molecular weight standards in kD. (C) VAEM images of PTENA, PTEND, and PI(3,5)P₂ binders fused to GFP demonstrate that only the fusion proteins that can functionally replace the For2A PTEN domain localize to spots at the cell cortex. In contrast, PTEND-GFP does not appear to have such a specific localization. See also Video 6. (D and E) Density of cortical For2A-GFP spots is reduced in cells overexpressing MTM1*-mCherry. (D) VAEM images of For2A-GFP in a wild-type cell or MTM1*-mCherry–overexpressing cell. (E) Quantification of cortical For2A-GFP density in wild-type or MTM1*-mCherry cells. Number of cells analyzed is: 4, wild type; 9, MTM1*-mCherry. Error bars represent standard SEM. Bars, 5 µm.