Fig. 3. IQGAP3 interacts directly with actin filaments. (A) Schematic representation of GST-IQGAP3. The structure of IQGAP3 and its various deletion mutants are represented. (B) GST-IQGAP3-WT or GST-IQGAP1-WT was mixed with F-actin, followed by incubation at room temperature for 1 hour. After the incubation, 50 µl of each reaction mixture was layered onto a 100-µl 20% (w/v) sucrose barrier and centrifuged. Two fifteenths of the supernatants or the pellets was subjected to SDS-PAGE, followed by immunoblot analysis with anti-GST antibody (top) and anti-actin antibody (bottom). One twentieth of the total proteins was shown as `input'. GST-IQGAP1-WT and GST-IQGAP3-WT were approximately 70% purity. The results shown are representative of three independent experiments. (C) The cosedimentation assay of the indicated IQGAP3 mutants with F-actin was performed as described in B. Two fifths of each pellet sample was subjected to SDS-PAGE. GST-RhoGDI was used as a negative control. The purity of GST-IQGAP3-CHD, -NR, and -N was 90%, 80% and 80%, respectively. The results shown are representative of three independent experiments.