Evaluation of different versions of γ-TuRC in the absence or presence of augmin. (A) TIRF microscopy images of MTs (red) grown in the presence of 10 nM indicated versions of γ-TuRC (green) together with (lower panels) or without (upper panels) 10 nM augmin-SNAP (blue). (B) Quantification of de novo nucleation of MTs from solution for experiments shown in upper panels of A. n = 3 experiments. (C) Western blot analysis of 0.1 pmol indicated versions of γ-TuRC-GFP as well as 0.2 and 0.4 pmol Strep-NEDD1^CDK1/PLK1 with γ-tubulin and NEDD1 antibodies. The amounts of NEDD1 in indicated versions of γ-TuRC-GFP were estimated by comparing their band intensities with those of defined quantities of NEDD1 proteins. (D) TIRF microscopy images of branching MT nucleation in the presence of 10 nM augmin^CDK1/PLK1-SNAP (blue) and 10 nM γ-TuRC^{ΔN/G3St/CDK1/PLK1}-GFP (green) together with Strep-NEDD1^CDK1/PLK1 (dark) at indicated concentrations. (E) Quantification of the total number of branched MTs per fan-like structure for experiments shown in D and lower panels of A. n = 3 experiments. (F) TIRF microscopy images of MTs (red) grown in the presence of γ-TuRC^{StN/CDK1/PLK1}-GFP at indicated concentrations together with (lower panels) or without (upper panels) 10 nM augmin^CDK1/PLK1-SNAP (blue). (G) Quantification of the overall γ-TuRC–dependent nucleation of MTs for experiments shown in F. n = 3 experiments. (H) Quantification of MTs originated from γ-TuRC in solution (left of dashed line) or lattice-bound pool of γ-TuRC (right of dashed line; branching nucleation) for experiments shown in F. n = 3 experiments. Scale bar, 2 μm. Data represent mean ± SD.