Formin-mediated actin assembly is inhibited by the introduction of Cap1/2 or CapZ. (A and B) Assembly of 1.5 μM rabbit muscle actin, 10% pyrene-labeled, was initiated in the presence of 7.5 μM yeast profilin (Pfy1) by the addition of 50 nM Bni1 (A) or 5 nM Bnr1 (B) and monitored by pyrene fluorescence enhancement. At 232 (A) or 145 s (B), yeast Cap1/2 or vertebrate CapZ at the indicated concentration was added to the reaction. Each curve is the average of three technical replicates. (C and D) Quantification of data in A and B. Actin assembly rate during a 230-s window 130 s after addition of Cap1/2 or CapZ. Rates are shown relative to the condition with formin only (Bni1 or Bnr1). Bars represent the mean and error bars indicate the SD. Statistical significance is calculated by two-way ANOVA with Tukey’s multiple comparisons test. Shown are the results from Tukey’s test (n.s., no significance; P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ****, P ≤ 0.0001). The two-way ANOVA shows no interaction between the row factor (Cap1/2 concentration) and column factor (formin used, Bni1 or Bnr1) and thus no significant difference between the two formins (P = 0.1043).