The newly identified motifs in TPX2 are necessary to stimulate branching MT nucleation. (A–H) Branching MT nucleation in Xenopus egg extracts and in the presence of 2 µM TPX2 α3–α7 or α5–α7, with and without various motif deletions. The same results were obtained with a lower TPX2 concentration of 0.75 µM. EB1-GFP (green) and Alexa Fluor 568–labeled porcine brain tubulin (red) were added to follow MT plus ends and MTs, respectively. Vanadate was added to prevent dynein-mediated gliding of MTs. All images were taken after 10 min. Brightness and contrast were adjusted for each image individually to optimize visual comparison of MT structures. Bar, 10 µm. See Video 2. (I and J) For each reaction, the number of individual MTs was counted for each time frame and then plotted against time. The data are displayed for TPX2 α3–α7 and TPX2 α5–α7 and their inactive deletion mutants. (K) Same as I and J, but here the number of MTs is averaged over two experimental replicates for TPX2 α5–α7 and the only active deletion mutant, TPX2 α5–α7 Δtrop. Error bars represent absolute error. All extract experiments were performed at least three different times.