Purified A1aY1 binder labels in vitro polymerized microtubules and fixed cellular microtubules. (A) Representative TIRF images of 5 µM A1aY1-GFP (green) bound to tyrosinated (magenta) and detyrosinated (cyan) microtubules with varying percentage of detyrosinated tubulin (CPA treated) incorporated during polymerization. Scale bars = 2 µm. (B) Mean ratio ± SEM of A1aY1-GFP fluorescence intensity bound to tyrosinated versus varying percentage of detyrosinated microtubules. Blue column bar represents pure tyrosinated microtubules. Data are derived from total 150 filaments analyzed for 100%, 75%, 50%, and 25% CPA microtubules (and 167 filaments from 0% CPA microtubules, n = 3) from n = 4 different experiments with two different batches of A1aY1 protein. (C) A1aY1-GFP staining and immunostaining of U2OS and H9C2 cells with anti-tyrosination and anti-detyrosination antibodies as indicated. Scale bars = 10 µm for the whole cell and 5 µm for the zoom panel. A.U., arbitrary units; MT, microtubule.