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. 2017 Jun 22;292(33):13566–13583. doi: 10.1074/jbc.M117.799247

Figure 3.

Figure 3.

Interactions of DAD-CT and DAD with actin. A, steady-state anisotropy of Alexa488NHS-labeled G-actin in the absence and presence of profilin as the function of DAD-CT, DAD, and DAD-CTArg–Ala concentrations, as indicated. Dashed lines in the corresponding colors show the fits to the data according to Equation 3. Dissociation equilibrium constants are summarized in Table 1. Error bars, standard deviations, n = 2–4. Final concentrations are as follows: [actin] = 0.2 μm; [LatA] = 4 μm; [profilin] = 0.8 μm; [NaCl] = 5 mm (circles); 17 mm (squares); 50 mm (diamonds). FL, fluorescently labeled protein. B, steady-state anisotropy of Alexa568NHS-labeled profilin in the presence of G-actin as the function of DAD-CT concentration, as indicated. Dashed line in the corresponding color shows the fit to the data according to Equation 3. Dissociation equilibrium constant is given in Table 1. Error bars, standard deviations, n = 2–3. Final concentrations are as follows: [actin] = 4 μm; [LatA] = 8 μm; [profilin] = 2 μm; [NaCl] = 5 mm. FL, fluorescently labeled protein. C, steady-state anisotropy of Alexa488NHS-labeled G-actin in complex with DAD-CT as a function of SALS-WH2 concentration. As controls, the steady-state anisotropies of G-actin in the absence of any binding proteins and G-actin saturated with SALS-WH2 (1.5 μm) are shown. Final concentrations are as follows: [actin] = 0.2 μm; [LatA] = 4 μm; [DAD-CT] = 20 μm; [NaCl] = 5 mm. FL, fluorescently labeled protein. D, representative polymerization kinetics of free G-actin and profilin/G-actin in the absence and presence of the C-terminal regions of DAAM, as indicated. Final concentrations are as follows: [actin] = 2 μm; [profilin] = 6 μm. E, TIRFM montages of actin assembly and representative skeletonized images showing the field of view of actin assembly in the absence and presence of DAD-CT, as indicated (for spontaneous actin assembly see Fig. 2D). Scale bar = 10 μm, time = min/s. Final concentrations are as follows: [actin] = 0.5 μm; [profilin] = 2 μm; [DAAM] = 42 μm. F, number of actin filaments nucleated spontaneously or in the presence of DAD-CT derived from skeletonized images. Final concentrations as in E, n = 16–20. G, elongation rate of individual actin filaments polymerized from free and profilin/G-actin in the absence and presence of DAD-CT (42 μm), DAD (42 μm), or DAD-CTArg–Ala (42 μm). Final concentrations as in E, n = 24–79. ns, not significant.