Fig. 2.

Like mammalian V-1, Dictyostelium V-1 binds CP tightly to prevent it from capping the barbed end. (A) SDS/PAGE gel showing the purified D.d. CP (lane 2) and GST-tagged D.d. V-1 (lane 3) used for the ITC measurement (lane 1, molecular mass markers). (B, Upper) The calorimetric titration of V-1 into CP, where 1.75 µl aliquots of 150 µM V-1 were injected into 10 µM CP at 20 °C. The experimental data points were fit to a 1:1 binding isotherm (error bars show the integration errors for each titration peak). (B, Lower) The fit residuals. (C) Representative pyrene-based, seed-initiated, actin polymerization assays in which increasing amounts of CP were added before seed initiation (see the color-coded key). (D) Mean and SDs from three independent assays performed as in C and plotted as the fraction of capped filaments vs. CP concentration. (E) Representative pyrene-based, seed-initiated, actin polymerization assays performed in the presence of 7 nM CP and increasing amounts of V-1 added before seed initiation (see color-coded key). (F) Mean and SDs from three independent assays performed as in E and plotted as the fraction of capped filaments vs. V-1 concentration.