Statplus

Each domain of CaM can be considered a two-site macromolecule and the affinities of Ca²⁺ binding to sites I and II or sites III and IV can be fit to a two-site Adair equation (Equation 3) (23 (link)) ${\overline{\mathrm{Y}}}_{\mathrm{t}\mathrm{o}\mathrm{t}\mathrm{a}\mathrm{l}}={\overline{\mathrm{Y}}}_2=\frac{{\mathrm{K}}_1\left[\mathrm{X}\right]+2{\mathrm{K}}_2{\left[\mathrm{X}\right]}^2}{2\left(1+{\mathrm{K}}_1\left[\mathrm{X}\right]+{\mathrm{K}}_2{\left[\mathrm{X}\right]}^2\right)}$ where K₁ (ΔG₁ = −RTlnK₁) is the sum of the intrinsic constants (k₁ + k₂) of the two sites in either domain of CaM, K₂ (ΔG₂ = −RTlnK₂) is the product of the intrinsic constants and the cooperativity constant (k₁·k₂·k_c), and [X] is the free [Ca²⁺].
To account for variations in intensity between experiments, the low and high endpoints of each titration were fit to the function [f(x)] shown in Equation 4 with nonlinear least squares analysis. $\mathrm{f}\left(\mathrm{X}\right)={\mathrm{Y}}_{\left[\mathrm{X}\right]\mathrm{l}\mathrm{o}\mathrm{w}}+{\overline{\mathrm{Y}}}_2\cdot \mathrm{S}\mathrm{p}\mathrm{a}\mathrm{n}$
The variable Y_[X]low corresponds to the intensity of the Ca²⁺-depleted sample, $\overline{\mathrm{Y}}$ ₂ is the average fractional saturation, and Span accounts for the difference in intensity at the highest and lowest ligand concentrations. Ca²⁺ titrations of CaM in the presence of the FGF12A_CaMBDp, FGF12_NTD, Na_V1.2_CTD, FGF12B and Na_V1.2_CTD, or FGF12A and Na_V1.2_CTD were analyzed assuming that each domain of CaM contained two functional Ca²⁺-binding sites. Pairwise comparisons of ΔG determinations were evaluated using the unpaired t test (GraphPad Prism, StatPlus); all p values were considered to be very (<0.005) or extremely (<0.0001) statistically significant unless noted otherwise.

Statistical analyses were performed with StatPlus and Prism5 (GraphPad, La Jolla, CA, United States). Data did not violate the assumption of normal distribution (Shapiro–Wilk test) and homogeneity of variance (Bartlett test). Therefore, the data were analyzed with parametric statistics: one-sample t-test or one-way analysis of variance followed by the planned pairwise multiple comparisons (Bonferroni). The significance level of statistical tests was set to 0.05.

Statistics were analysed with Microsoft Excel and AnalySoft Statplus, Graphpad Prism 6 or SPSS on the number of samples indicated. F-test was used to determine equivalence of variance and the appropriate Student's t-test or ANOVA with Scheffé post hoc test applied unless otherwise stated. All graphs show mean and standard error of the mean. Numbers on columns represent number of fish scored.