Fig. 6. Actin polymerization regulates the interaction between cortactin and dynamin. (A) Purified GST-Dyn-PRD (30 nM) was mixed with immobilized His-cortactin ranging from 0-200 nM in actin polymerization buffer plus G-actin (2 µM) and Arp2/3 complex (200 nM), and incubated at room temperature for 30 minutes. As a control, the reaction was also performed in the same buffer without G-actin and Arp2/3 complex. The samples were briefly centrifuged and the supernatants were analyzed for the presence of remaining GST-Dyn-PRD by immunoblotting with GST antibody. Amounts of GST-Dyn-PRD on the blot were quantified by digital scanning and normalized to the percentage of depletion. The resulting data were used to fit a rectangular hyperbola, yielding apparent K_d values as indicated. Inset, showing that GST has little binding activity to cortactin as compared to GST-Dyn-PRD. (B) The interaction of GST-Dyn-PRD and cortactin was carried out with G-actin plus Arp2/3 complex or G-actin without Arp2/3 complex. Cortactin was also preincubated with G-actin and Arp2/3 complex for 20 minutes prior to incubation with GST-Dyn-PRD for additional 20 minutes.