Figure 2.

tau mutants at the Pro-301 residue significantly impaired MT binding. A and B, cell-based MT-binding assay performed with HEK293T cells transfected to express WT tau with or without the presence of paclitaxel as described under “Experimental procedures.” Antibody specific for β-tubulin (clone TUB 2.1) was used to assay the polymerization of tubulins. 3026 is a polyclonal antibody against total tau. S = supernatants; P = pellet fractions. Without paclitaxel, the majority of tubulin is not polymerized and soluble, whereas with paclitaxel, the majority of tubulin is polymerized as MTs in the pellet fraction. C, P301L, P301S, and P301T tau mutants are in the PGGG motif of the R2 repeat. In the presence of paclitaxel, P301L (D), P301S (E), and P301T (F) all demonstrate significantly decreased MT binding when compared with WT tau. The relative molecular masses of protein markers are indicated on the left. On the right, FL is for full-length tau, and the brace indicates degradative tau bands. G, one-way ANOVA with Dunnett's test was performed with n = 18 for WT tau and n = 3 for each of these tau mutants. ****, p < 0.0001.