Purified protein gels and quantitative Western blots. (A) Coomassie staining of purified protein size separated via SDS-PAGE. (B) Quantification of three or four independent measurements for endogenous actin regulators within the HSS extracts. Black horizontal lines indicate the measurement mean. (C–G) Example blots of purified protein and X. laevis egg HSS extracts used to calculate concentration of proteins via quantitative Western blotting, probed as TOCA-1 (C), N-WASP (D), Ena (E), VASP (F; where we combined the bands suspecting they represented different phosphorylation states), and Fascin (G). (H) Purified IRSp53. (I) Time course showing that Alexa488-SNAP-IRSp53 foci do not localize to FLS actin bundles, whereas control N-WASP does (both added at 50 nM). Scale bars = 1 μm. (J) Alexa488-SNAP-IRSp53 binds the supported lipid bilayer. Scale bars = 10 μm. HSS, high-speed supernatant. TIRFM, total internal reflection fluorescence microscopy.