Changes in microtubule binding of tau isoforms when phosphorylated with GSK-3β. Microtubule binding reactions were performed at varying concentrations of tau (0 μM to 10 μM) and bound vs. free tau (in μM) for both unphosphorylated (black circle) and phosphorylated (red circle) was plotted (A-F). Three separate trials are plotted together to demonstrate the variability in the reactions. The data were fit to a nonlinear single site binding equation for unphosphorylated (black solid line) and phosphorylated (red solid line) tau isoforms (A-F). The 95% confidence intervals of the fits are drawn as dashed lines. Outliers existed for phosphorylated samples, are shown as red open boxes, and were excluded from calculations. (G) The dissociation constant (Kd) (in μM) was determined for unphosphorylated (white bars) and phosphorylated (red bars) isoforms by Kd = the concentration of tau required to reach 1/2 Bmax. Data represents the average of 3 trials ± SEM.