Properties of APC as an actin assembly–promoting factor. (A) Comparison of nucleation effects for 20 nM APC-B and 20 nM C-mDia1 at different concentrations of actin monomers (5% pyrene labeled). (B and C) Effects of 2 nM C-mDia1 or 20 nM APC-B on the assembly of actin (2 µM; 5% pyrene labeled) in the presence or absence of 3 µM profilin. (D) Increase in barbed end elongation rate of individual actin filaments by C-mDia1 and profilin but not by APC-B. Rates of elongation were measured in real time by TIRF microscopy and averaged for >10 filaments. Error bars, standard deviation. (E) Native PAGE assay for APC-B binding to G-actin. Reactions were loaded on gels and run toward either the cathode (top) or anode (bottom), then gels were Coomassie stained. (F) Fluorescence-based assay for concentration-dependent binding of APC-B (0–75 nM) to G-actin (100 nM; 100% pyrene labeled). Dashed line indicates binding saturation at a 1:2 molar ratio of APC-B to actin. Error bars, standard deviation (n = 3). (G) 5 µM profilin does not affect the ability of 50 nM APC-B to increase the fluorescence of pyrene–G-actin (0.1 µM, 100% labeled). Error bars, standard deviation (n = 3).