Comparison of TR-F and cosedimentation actin-binding assays for C0–C2 mutants. Unphosphorylated and phosphorylated WT and five mutants of C0–C2 were assayed using IAEDANS–actin or IAEDANS–actin–Tm. (A) TR-F using 1.25 µM C0–C2. (B) Cosedimentation using 1.25 µM C0–C2. (C) TR-F using 10 µM C0–C2. (D) Cosedimentation using 10 µM C0–C2. *, P < 0.05 for comparisons with WT under the same conditions at substoichiometric binding levels (1.25 µM C0–C2, A and B). In A and B, reduction in binding due to phosphorylation was significant (P < 0.005) in all cases with the exception of EASE C0–C2 binding to actin–Tm measured by TR-F. Data are provided as mean ± SE (n > 4).