PI(3,5)P₂ regulates the interaction of cortactin with actin filaments. (A) Schematic of WT and mutant cortactin constructs. (B, left) Representative Western blot from n = 3 Arp2/3 binding experiments, probed with an antibody to Arp2. (right) Coomassie-stained gel of GST-tagged proteins immobilized on glutathione beads used in Arp2/3 binding experiment. The same amount of beads was loaded in each lane and used in the experiment. (C and D) Representative Western blots from (C) GST-N-term and (D) GST-Δ4RP cortactin–pull-down experiments. Cortactin proteins bound to PI(3,5)P₂-liposomes were identified with an anti-GST antibody. Relative binding affinity was quantified by densitometric analysis of Western blot data from three independent experiments. Mean ± SE. *, P < 0.05; ****, P < 0.0001. (E and F) F-actin competes with PI(3,5)P₂ for binding to cortactin. (E) Increasing concentrations of actin filaments were incubated with 70 nM cortactin and 250 nM PI(3,5)P₂-containing liposomes. In the presence of F-actin, cortactin binding to liposomes was significantly reduced. (F) Data points show mean binding from four independent experiments. Fit to hyperbolic decay model yields a K_i value of 0.461 µM.