VASP antagonizes capping protein barbed end association in the presence of profilin–actin. (A) Actin filament length distribution after 5 min of barbed end growth in the presence of 2 µM Mg-ATP-actin (10% Alexa Fluor 488), 2 µM hPro1, 0–20 nM MmCP, and 0–50 nM VASP. Bar, 10 µm. (B) Average number of actin subunits delivered to barbed end before termination of growth by MmCP. The dash line represents the predicted actin filament length if the filaments had been elongating 2.4 times faster than 2 µM profilin–actin alone (n ≥ 100 filaments). (C) Rate of actin filament capping in the presence of 2 µM Mg-ATP-actin (10% Alexa Fluor 488), 2 µM hPro1, 2–4 nM MmCP, and 0–50 nM VASP. Calculation of K_obs was achieved by fitting data to the [BE]_free * exp^(−-k/τ₁) equation in KaleidaGraph (n ≥ 150 filaments tracked). (D) Plot of K_obs (sec⁻¹) versus MmCP concentration. The slope gave a capping protein association rate constant of 12 µM⁻¹ sec⁻¹ and 1.9 µM⁻¹ sec⁻¹, in the absence or presence of 50 nM VASP, respectively.