(A) A control immunodepletion of the extract with total IgG fraction antibodies still produced fan-shaped structures composed of branching microtubule and did not significantly affect levels of augmin (Dgt4 subunit), γ-TB and TPX2 levels as quantified in a Western blot (bottom, error bars represent s.d. of three independent experiments with different extracts). (B) Immunodepletion of γ-TB (bottom) almost extinguished microtubule nucleation; most fields had no or a few microtubules; after longer times (7 min), rare examples of fan-shaped structures with few, but long microtubules could be found on the coverslip (see insert). (C) Immunodepletion of the augmin Dgt4 subunit, which depletes the entire augmin complex and one third of γ-TB (bottom diagram), abolished branching microtubule nucleation, but still allowed for microtubule growth. (D) Similarly, immunodepletion of TPX2 abolished branching microtubule nucleation, but still allowed for microtubule growth. Scale bars, 10 μm. See The time shown indicates the approximate time of recording after adding the extracts to the flow cell. for sequences of these immunodepletions. See also . (E) Quantification of microtubule number over time for different immunodepletions. Reactions were made in the presence of RanQ69L plus TPX2 (except for the TPX2 depletion, in which case only RanQ69L was added).