Cortactin directly interacts with PI(3,5)P₂. (A) The affinity of full-length cortactin was examined using a protein-lipid overlay assay. The left panel indicates the identity of lipid species on the PIP strip. Cortactin binds most strongly to PI(3,5)P₂. Binding of the FYVE domain of Hrs (2xFYVE) and PH domain of PLC-δ1 to PI(3)P and PI(4,5)P₂, respectively, served as positive controls. (B) Pull-down of GST–cortactin by liposomes bearing the indicated PIPs. GST-2xFYVE, and GST alone are respectively positive and negative controls. Western blots were probed with anti-GST antibody. (C) The relative binding of GST-fusion proteins to liposomes containing different PIPs was quantified by densitometric analysis of GST immunoblots from three independent experiments, except for positive control GST-2xFYVE that was tested once for pull-down by PI(3)P. Mean ± SE. **, P < 0.01. (D) Cortactin binding PI(3,5)P₂- or PI(5)P-containing liposomes, plotted as a function of PIP concentration. Data points in the PI(3,5)P₂ and negative control PI(5)P binding curves represent means calculated from data points of five and two different experiments, respectively. The K_d for cortactin–PI(3,5)P₂ interaction obtained from nonlinear regression of the data are 30 nM.