TR-F binding assays for actin and actin–Tm compared with cosedimentation. Unphosphorylated C0–C2 (solid lines) and phosphorylated C0–C2 (dotted lines) were assayed. (A) TR-F using IAEDANS–actin (thin lines). (B) TR-F using IAEDANS–actin–Tm (thick lines). (C) TR-F using IAEDANS–actin and IAEDANS–actin–Tm showing 0–5 µM C0–C2 added. (D) Cosedimentation using actin. (E) Cosedimentation using actin–Tm. (F) Cosedimentation using IAEDANS–actin and IAEDANS–actin–Tm showing 0–5 µM C0–C2 added. Arrows in A–F indicate the C0–C2 concentrations to be used in future screens (thin arrows, 2.5 µM for actin alone; thick arrows, 1.25 µM for actin–Tm). (G) Linear correlation plot of unphosphorylated C0–C2 binding measured by TR-F (change in lifetime) and cosedimentation ([bound C0–C2]/[actin]) for actin (R² = 0.96) with each assay readout normalized to 1 (at 20 µM C0–C2). (H) Linear correlation plot in the presence of Tm, under conditions of G, for unphosphorylated C0–C2 binding to actin–Tm (R² = 0.87). Refer to Table 1 for statistical analysis of fitted binding properties for curves. Data are provided as mean ± SE (n > 4).