Fig. 4. Increase in association of Arp2/3 with actin filaments by wild-type WASP family proteins. Association of Arp2/3 (0.25 µM) with actin filaments (2 µM) in the presence or absence of N-WASP, its mutants or WAVE2 (1 µM) was analyzed by co-sedimentation. Filaments were made by polymerizing G-actin. Phalloidin filaments were made by adding phalloidin after 1 hour of polymerization. Filaments and phalloidin filaments are composed of ATP actin caps of <100 actin molecules (<0.6 µm per 5 µm (average) filament) and ADP filaments. ADP-Pi/phalloidin filaments were made by polymerizing G-actin with phalloidin. Co-sedimented Arp2/3 complex was monitored by western blotting with anti-Arp3 antibody (a). The extent of association at 2 µM of F-actin was quantified by densitometry and expressed as the fold increase in comparison with Arp2/3 only (b). Bars show the standard deviations.
