For2A PTEN domain and PTENA specifically bind PI(3,5)P₂. (A) Representative lipid-overlay assay shows that the For2A PTEN-GST binds broadly to phosphoinositides, with highest specificity to PI(3,5)P₂. PTENA binds PI(3,5)P₂ strongly. PTEND also binds PI(3,5)P₂, but weaker than PTENA. Additionally, PTEND also binds PI(3)P. GST alone does not interact with phosphoinositides. The numbers indicate amount in picomoles of spotted phosphoinositide. At least three independent experiments were performed for each overlay assay. (B) Immunoblot of For2A PTEN-GST, GST-PTENA, and GST-PTEND using an anti-GST antibody shows that For2A PTEN-GST and GST-PTENA preferentially bind to beads covalently linked to PI(3,5)P₂ as compared with control beads. In contrast, GST-PTEND binds similarly to control and PI(3,5)P₂ beads. Numbers are molecular weight standards in kD. (C) Immunoblot of For2A PTEN-GST and GST-PTENA using an anti-GST antibody demonstrates that binding to PI(3,5)P₂ beads is specifically displaced in the presence of exogenous PI(3,5)P₂, but not other phosphoinositides. Numbers are molecular weight standards in kD.