CDK1- and PLK1-dependent phosphorylation is crucial for augmin–γ-TuRC interaction and hence for branching MT nucleation. (A and B) StrepTactin pull-down assay with extracts of nocodazole-arrested mitotic HAUS8-GFP-Strep knock-in HeLa cells treated with CDK1 inhibitor RO3306 (A) or PLK1 inhibitor BI2536 (B) for indicated time periods, analyzed by Western blotting with indicated antibodies. (C and D) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing augmin-GFP-Bio (bait) and γ-TuRC-GFP (prey), treated with (+) or without (−) RO3306, BI2536, or both (C); or together with (+) or without (−) untagged CDK1/cyclin B, HA-PLK1, or both (D), analyzed by Western blotting with indicated antibodies. (E) TIRF microscopy images of branching MT nucleation in the presence of recombinant complete augmin–γ-TuRC complex (10 nM augmin) copurified from nocodazole-arrested mitotic HEK293T cells expressing augmin-mCherry-Strep and γ-TuRC-GFP, together with or without untagged CDK1/cyclin B, HA-PLK1, or both. The concentrations of γ-TuRC were 0.67, 0.74, 0.75, and 1.0 nM for augmin–γ-TuRC, augmin–γ-TuRC^PLK1, augmin–γ-TuRC^CDK1, and augmin–γ-TuRC^CDK1/PLK1, respectively. Images were acquired 15 min after imaging. (F) Quantification of the total number of branched MTs per fan-like structure at indicated time points in the assays performed with indicated complexes. n = 3 experiments. (G) Quantification of intensities of augmin-mCherry and γ-TuRC-GFP along MTs for the experiments shown in E. The values were normalized to the intensity of the augmin–γ-TuRC^CDK1/PLK1 complex. n = 30 MTs from three experiments. Scale bar, 2 μm. Data represent mean ± SD.