PSD-95 decreases the lifetime of EB3 comets in hippocampal neurons. A, Diagram of a mutant form of EB3 with key amino acids of the proline-rich region changed to threonines. B, Wild-type, but not EB3-THR, coimmunoprecipitates with PSD-95 from extracts of COS-7 cells coexpressing the indicated proteins. The anti-EB3 antibody used for Western blotting recognizes a nonspecific (ns) ∼50 kDa protein band in COS-7 cell lysates. IP, Immunoprecipitation; WB, Western blot. C, Low-magnification images of GFP-tagged EB3 comets in a growing neurite. Asterisks indicate representative comets. D, Representative time series images of GFP-tagged EB3 and EB3-THR comets in the dendrites of hippocampal neurons and GFP-tagged EB3 and EB3-THR comets in hippocampal neurons also expressing PSD-95-mRFP or PSD-95ΔSH3-mRFP. Images were taken at 18–24 h posttransfection, which was performed at the time of plating. Scale bars: 1 μm. Arrows indicate first and last appearances of comets. Asterisks track with tip of the comets. E, Quantitation of the effect of PSD-95-mRFP overexpression on EB3-GFP and EB3-THR-GFP comet lifetime. Overexpression of PSD-95-mRFP decreases the lifetime of EB3-GFP comets but does not decrease the lifetime of EB3-THR-GFP comets. *p < 0.05; **p < 0.01 by Kruskal–Wallis test followed by Dunn's multiple comparison test compared to EB3-GFP. n ≥ 15 comets for each condition. F, Quantitation of the effect of PSD-95 overexpression on EB3-GFP and EB3-THR-GFP comet velocity. Overexpression of PSD-95-GFP decreases the velocity of both EB3-GFP and EB3-THR-GFP comets, suggesting that this effect is not specific to the direct interaction between PSD-95 and EB3. *p < 0.05; **p < 0.01 by Kruskal–Wallis test followed by Dunn's multiple comparison test compared to EB3-GFP. n ≥ 15 comets for each condition.