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Figure 3.
Inhibition of actin polymerization by the ACD induced
cross-linking. (A–C) Polymerization and cross-linking of 10
μM rabbit skeletal actin in the presence or absence of ACD were
initiated simultaneously by adding 1.0 mM MgCl[2] and 50 mM KCl
and monitored by light scattering (A), sedimentation assay (B),
and electron microscopy (C). Compared with polymerization of
actin in the absence of ACD, the polymerization was strongly
inhibited at 1:1000 mole ratio of ACD to actin and it was
blocked completely at 1:100 mole ratio to actin. ACD
cross-linked actin oligomers form aggregates (C), which do not
pellet during ultracentrifugation (B). In all cases, s and p
designate supernatant and pellet fractions, respectively.
(D–F) The increase in light scattering upon addition of 15 μM
phalloidin (red trace) or 10 μM cofilin (blue trace) indicates
that the polymerization of ACD cross-linked actin oligomers (at
1:100 mole ratio of ACD to actin) can be rescued by these
agents. To estimate the extent of polymerization and the
appearance of the filaments, samples from (D) were analyzed by a
sedimentation assay (E) and electron microscopy (F).
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