Figure 6.
Biochemical characterization of phosphorylation-dependent NEDD1-augmin interaction and branching MT nucleation. (A) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing HA-NEDD1 (prey) together with Bio-GFP–tagged augmin holo complex, H1/H4/H3/H5 subcomplex, or eight individual subunits (bait), analyzed by Western blotting with indicated antibodies. (B) Schematic overview of the domain organization of NEDD1 and the deletion mutants, and summary of their interactions with augmin. N, N-terminus; C, C-terminus. (C) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing Bio-GFP–tagged augmin holo complex (bait) together with HA-tagged NEDD1 or its indicated fragments (prey), analyzed by Western blotting with indicated antibodies. Asterisk indicates the bands of HA-BirA, which was cotransfected for biotinylating bait proteins. (D and E) Coomassie blue–stained gels with flag-NEDD1 and augmin-GFP-Strep purified from nocodazole-arrested mitotic HEK293T cells in the presence of either coexpressed untagged CDK1/cyclin B, HA-PLK1, or individual inhibitors. Asterisks indicate endogenous biotinylated proteins that were captured by StrepTactin beads during purification. (F and G) StrepTactin pull-down assays using indicated versions of augmin-GFP-Strep immobilized on StrepTactin beads as the bait and indicated versions of flag-NEDD1 eluates as the prey, analyzed by Western blotting with indicated antibodies. (H) TIRF microscopy images of branching MT nucleation in the presence of indicated pairs of 10 nM augmin-SNAP (blue) and 10 nM γ-TuRC-GFPStN (green). Images were acquired 10 min after imaging. (I) Quantification of the total number of branched MTs per fan-like structure at indicated time points in the assays performed with indicated pairs of complexes. n = 3 experiments. (J and K) Quantification of intensities of augmin-SNAP (J) and γ-TuRC-GFPStN (K) along MTs for the experiments shown in H. The values were normalized to the intensity of the pair of augminCDK1/PLK1 and γ-TuRCStN/CDK1/PLK1. n = 50 MTs from three experiments. Scale bars, 2 μm. Data represent mean ± SD. ***, P < 0.001, two-tailed t test.

Biochemical characterization of phosphorylation-dependent NEDD1-augmin interaction and branching MT nucleation. (A) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing HA-NEDD1 (prey) together with Bio-GFP–tagged augmin holo complex, H1/H4/H3/H5 subcomplex, or eight individual subunits (bait), analyzed by Western blotting with indicated antibodies. (B) Schematic overview of the domain organization of NEDD1 and the deletion mutants, and summary of their interactions with augmin. N, N-terminus; C, C-terminus. (C) Streptavidin pull-down assay with extracts of nocodazole-arrested mitotic HEK293T cells expressing Bio-GFP–tagged augmin holo complex (bait) together with HA-tagged NEDD1 or its indicated fragments (prey), analyzed by Western blotting with indicated antibodies. Asterisk indicates the bands of HA-BirA, which was cotransfected for biotinylating bait proteins. (D and E) Coomassie blue–stained gels with flag-NEDD1 and augmin-GFP-Strep purified from nocodazole-arrested mitotic HEK293T cells in the presence of either coexpressed untagged CDK1/cyclin B, HA-PLK1, or individual inhibitors. Asterisks indicate endogenous biotinylated proteins that were captured by StrepTactin beads during purification. (F and G) StrepTactin pull-down assays using indicated versions of augmin-GFP-Strep immobilized on StrepTactin beads as the bait and indicated versions of flag-NEDD1 eluates as the prey, analyzed by Western blotting with indicated antibodies. (H) TIRF microscopy images of branching MT nucleation in the presence of indicated pairs of 10 nM augmin-SNAP (blue) and 10 nM γ-TuRC-GFPStN (green). Images were acquired 10 min after imaging. (I) Quantification of the total number of branched MTs per fan-like structure at indicated time points in the assays performed with indicated pairs of complexes. n = 3 experiments. (J and K) Quantification of intensities of augmin-SNAP (J) and γ-TuRC-GFPStN (K) along MTs for the experiments shown in H. The values were normalized to the intensity of the pair of augminCDK1/PLK1 and γ-TuRCStN/CDK1/PLK1. n = 50 MTs from three experiments. Scale bars, 2 μm. Data represent mean ± SD. ***, P < 0.001, two-tailed t test.

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