Fig. 2

Cofilin displaces tau from tubulin/microtubules and inhibits tau-induced microtubule assembly. a, b In vitro tubulin–cofilin binding assay using recombinant proteins (His-tau, cofilin, tubulin) at indicated amounts, showing that tau reduces the tubulin–cofilin complex in a dose-dependent manner. b Quantification of tubulin–cofilin complexes. Data are expressed as mean ± SEM (one-way ANOVA with Tukey post hoc, n = 4, ***p = 0.0003, ^#p < 0.0001). c, d In vitro tau–tubulin binding assay using recombinant proteins (His-tau, tubulin, and cofilin) at indicated amounts, showing that cofilin inhibits the tau–tubulin complex in a dose-dependent manner. d Quantification of tubulin–tau complexes. Data are expressed as mean ± SEM (one-way ANOVA with Tukey post hoc, n = 4, **p = 0.0073, ***p = 0.0002). e Binding of cofilin and tau to microtubules were performed by microtubule-binding sedimentation assay. Indicated amounts of recombinant cofilin and/or 2 µg recombinant tau were incubated with or without 0.4 nM pre-polymerized microtubules, and microtubule-associated proteins were monitored by co-sedimentation and subsequent SDS-PAGE analysis. Representative western blots showing reduced microtubule-associated tau and increased supernatant tau by cofilin in vitro. f Quantification of supernatant and microtubule-associated pelleted tau with indicated amounts of cofilin. Data are expressed as mean ± SEM (one-way ANOVA with Tukey post hoc, n = 6, supernatant tau *p = 0.0365, ^#p < 0.0001, pellet tau *p = 0.0498, **p = 0.0014). g Tubulin polymerization was measured by turbidity at 340 nm in the presence of indicated recombinant proteins (2 μg) (two-way repeated measures ANOVA, ^#p < 0.0001, n = 4/condition)