The astrin-kinastrin/SKAP complex localizes to MT plus ends. (A) GFP–astrin cells were imaged every 1.25 s. The first, third, fifth, and seventh frames were projected into one image using different colors as indicated. (B) Methanol-fixed GFP–astrin cells were stained for EB1 and GFP. (C) GFP–astrin cells transiently expressing EB1-mCherry were imaged every 3.2 s. The arrowheads indicate one growing MT plus end that can be followed through the different frames of the movies. (D) Methanol-fixed HeLa cells were stained for astrin and EB1. Single focal planes are shown. (E) GFP–astrin cells were mock-treated or treated with 100 ng/ml nocodazole for 2 h, then stained with CREST antiserum and for EB1 and GFP. (F) GFP–astrin cells were treated with control or siKinastrin oligos for 72 h, then stained for EB1 and kinastrin. Boxed regions are shown enlarged on the right. (G) MT polymerization in the presence of buffer, taxol, GST, or GST–kinastrin at the indicated concentrations was followed in a fluorimeter. Each curve is representative of three independent experiments. (H) HeLa cells were treated overnight with 200 µM monastrol followed by 1 h cold treatment on ice. MT regrowth was initiated by the addition of fresh 37°C medium then monitored for 45 min by staining for astrin, tubulin, and DNA (DAPI). (I) HeLa cells treated with control, Nuf2, or kinastrin siRNA oligos for 72 h were cold treated for 10 min to visualize stable kinetochore fibers. The bottom row shows enlarged views of the boxed regions. Bars, 10 µm unless otherwise indicated.