The GMPCPP-stabilized MTs were assembled using 50 µM unlabeled porcine tubulin containing 1:12 biotin-labeled porcine tubulin (Cytoskeleton Inc.) and polymerized in the presence of 1 mM GMPCPP (Jena Bioscience) at 37 °C for 30 min. Approximately 300 nM GMPCPP-stabilized MTs were introduced into a flow chamber and allowed to bind to a 20% antibiotin antibody (clone BN-34, MilliporeSigma) for 10 min. Then, 100 nM of the specified SPR2-GFP proteins along with 2.5 µM 1:9 rhodamine-labeled porcine tubulin were introduced into the flow chamber in BRB80 buffer containing 1% methyl cellulose (4,000 cP, MilliporeSigma), 50 mM DTT, and an oxygen-scavenging system (Fan et al. 2018 (link)). GFP and rhodamine were excited using 5 mW 488 and 561 nm diode-pumped solid-state lasers, and the images were collected by a 100× (NA 1.45) objective and a back-illuminated electron-multiplying CCD camera (Image EM; Hamamatsu) at 2 s intervals. The extent of colocalization between SPR2-GFP and soluble rhodamine-tubulin was analyzed using the JACoP plugin (Bolte and Cordelieres 2006 (link)).