Reconstituted Xenopus augmin recovers branching MT nucleation that was impaired by depletion of endogenous augmin in Xenopus egg extracts. (A) The eight-subunit complex, augmin was stoichiometrically reconstituted in vitro, confirmed by size-exclusion chromatography using Superdex 200 increase 10/300 GL column. The calibrated void volume of the column is 8.73 ml. Fractions (1–6) were loaded to a SDS-PAGE gel (4–12% gradient), visualized by silver staining (right). (B) ID of endogenous augmin was performed using IgG as a control and anti-H1 antibody. Branching MT nucleation was activated by RanQ69L, in which Cy5-labeled tubulin and mCherry-labeled end-binding protein 1 (EB1-mCherry) highlight MTs and growing MT plus ends, respectively, pseudocolored in red and green. Bar, 10 µm. Recombinant augmin was added to the immunodepleted extract (Δ H1) at increasing concentrations, demonstrating that branching MT nucleation is restored in a concentration-dependent manner. Augmin localizes along the length of MTs, visualized by GFP in gray (bottom panel). Images were taken 35 min after sample preparation. (C) Anti-H1 antibody stoichiometrically depletes augmin subunits and add-back of 150 nM recombinant augmin to the H1-ID extract can be compared with the endogenous level (IgG). The fold changes for each subunit relative to the endogenous levels are depicted. These numbers are not absolute, because each antibody recognizes its antigen with different specificities.