Figure S2.
Buffer conditions optimization of the TR-F assay for C0–C2 binding to IAEDANS–actin and IAEDANS–actin–Tm. Reduction in lifetimes (% decrease) for either 1 µM IAEDANS-labeled actin or actin–Tm binding to the indicated concentrations of C0–C2 that was not phosphorylated (gray and black) or phosphorylated by PKA (+PKA, pink and red). Note the first bar left of the six colored bars in each group shows 0% decrease in lifetime with baseline error bars for actin, or actin–Tm, alone (i.e., 0% decrease relative to itself). (A) Binding buffers at varying pH and either 100 mM KCl or NaCl. (B) Three concentrations of KCl in binding buffer. (C) Different ratios of actin/Tm. In C, for unphosphorylated C0–C2, significant differences comparing actin alone to actin/Tm are indicated ($,P < 0.05). In A–C, significant differences between minus and plus PKA are indicated (*, P < 0.05; #, P < 0.005). Data are provided as mean ± SE (n > 4).

Buffer conditions optimization of the TR-F assay for C0–C2 binding to IAEDANS–actin and IAEDANS–actin–Tm. Reduction in lifetimes (% decrease) for either 1 µM IAEDANS-labeled actin or actin–Tm binding to the indicated concentrations of C0–C2 that was not phosphorylated (gray and black) or phosphorylated by PKA (+PKA, pink and red). Note the first bar left of the six colored bars in each group shows 0% decrease in lifetime with baseline error bars for actin, or actin–Tm, alone (i.e., 0% decrease relative to itself). (A) Binding buffers at varying pH and either 100 mM KCl or NaCl. (B) Three concentrations of KCl in binding buffer. (C) Different ratios of actin/Tm. In C, for unphosphorylated C0–C2, significant differences comparing actin alone to actin/Tm are indicated ($,P < 0.05). In A–C, significant differences between minus and plus PKA are indicated (*, P < 0.05; #, P < 0.005). Data are provided as mean ± SE (n > 4).

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