Fig. S2.

Like mouse V-1, D.d. V-1 cannot uncap CP-capped filaments and is readily displaced from CP by the CP binding domain of D.d. CARMIL. (A) Representative pyrene-based, seed-initiated, actin polymerization assays performed in the presence of 7 nM CP and increasing amounts of GST-V-1 added 20 s after seed initiation to detect the uncapping of CP-capped filaments (see color-coded key). No uncapping (i.e., restoration of polymerization) was observed even at the highest concentration of GST-V-1 tested (2 µM). (B) Shown is a Coomassie Blue-stained gel (inverted image) of a set of complex exchange reactions (26), in which resin coated with GST-V-1 was saturated with CP to create the CP:V-1 complex, washed, incubated for 10 min with the isolated CAH3 domain of D.d. CARMIL at the indicated molar ratios, and then fractionated into supernatant and pellet. The positions of GST-V-1, CPα, CPβ, and CAH3 are indicated. The CAH3 domain can be seen to drive in a dose dependent manner the dissociation of CP from GST-V-1, as evidenced by the progressive shift of CP from the pellet to the supernatant as more CAH3 is added.