PSD-95 decreases the binding of EB3 to microtubules in heterologous cells and inhibits microtubule assembly in a cell-free system. A, Microtubules were immunoprecipitated from lysates of COS-7 cells expressing EB3-GFP and exogenously added GST-PSD-95 or GST-PSD-95ΔSH3. Immunoprecipitates were subjected to SDS-PAGE and Western blotting for the presence of EB3. B, Light scattering of microtubules at OD₃₅₅ in the presence of 1 mm GTP was used to detect the effect of PSD-95 on microtubule polymerization. Addition of 4 m GST-PSD-95, but not GST-PSD-95ΔSH3, slows the polymerization of purified tubulin compared to the addition of equimolar concentrations of GST alone. Inset, Western blots of purified tubulin fractions (right lane), used in the above polymerization assay, show detectable amounts of EB3. The immunoreactive protein band comigrates with that from a rat brain homogenate (left lane). C, Quantification of total tubulin polymerization shows that GST-PSD-95, but not GST-PSD-95ΔSH3, lowers the total amount of polymerization that occurred as measured by V_max of the curve. *p < 0.05 by two-way repeated measures ANOVA followed by a Bonferroni multiple comparisons test to compare each point on the curve. n = 3. Error bars indicate SEM.