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Fig. 2. The first actin-binding domain of T-plastin (ABD1), which binds but does not bundle actin filaments, has a similar effect on bead movement to wild-type T-plastin. (A) Protein domain scheme of T-plastin and ABD1 variant. (B) Cosedimentation of T-plastin or the ABD1 variant with F-actin in vitro. G-Actin was polymerized in the presence of T-plastin (+ T-plastin) or ABD1 (+ ABD1), or their absence (control). The molar ratio of actin:plastin or actin:ABD1 was 4:1. Proteins were centrifuged at high speed to sediment actin filaments (top) or at low speed to sediment actin bundles (bottom). Proteins in supernatants (S) and pellets (P) were separated by SDS-PAGE under reducing conditions. Protein bands were stained with Coomassie Brilliant Blue. (C) Time-lapse phase-contrast microscopy of bead movement. Beads were incubated in HeLa extracts supplemented with 1.1 µM or 2.2 µM ABD1. No depolymerization of the actin tail is observed when ABD1 is added (black arrow). (D) Bead velocity as a function of ABD1 concentration Bead velocity shows a bell-shaped dependence on ABD1 concentration, with a maximum velocity for 1.1 µM ABD1. Each bar represents the mean±s.d. of ten measurements. An asterisk (*) indicates results that differ significantly from the result obtained with the addition of 1.1 µM ABD1 (P<0.0006).
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