Fig. 2. F-actin binding region of N-WASP/WAVE2. (a) The association of isolated basic region with filaments was analyzed by co-sedimentation assay. The amino acid numbers of these fragments from N-WASP or WAVE2 are shown. Association was analyzed by western blotting to either glutathione-S-transferase tag or His tag. (b) Dose dependence of actin filaments (which are composed mostly of ADP filaments and small amounts of filaments with ATP and ADP-Pi caps) against association with the isolated basic region by co-sedimentation assay. The association was monitored by western blotting followed by densitometry. (c) Comparison of the affinity of isolated basic region against ADP-Pi or ADP filaments by co-sedimentation assay. Association was analyzed by western blotting. ADP-Pi filaments were made by polymerizing actin in the presence of phalloidin. ADP filaments were made by hexokinase treatment. To estimate the effect of phalloidin staining, some of the ADP filaments were mixed with phalloidin and also used in the assays.
