Figure 3.
SEPT9 provides a molecular link between dynein and dynactin through its GTP-binding and NTE domains, respectively.(A) Coomassie-stained gel shows the recombinant NTE and G domains of SEPT9, which were used in pull-down assays with purified dynein and dynactin. Pull-downs were Western blotted with antibodies against DHC and p150GLUED. Bar graph shows the mean ratio of DHC to NTE or G domain and p150GLUED to NTE or G domain fragments of His-SEPT9 intensities from three independent experiments; error bars indicate SEM. Schematic depicts the domains of SEPT9 and the position of the catalytic T339 residue required for GTPase activity. (B) Coomassie-stained gel shows the results of protein binding assays between recombinant His-SEPT9 and GST (control) or the GST-tagged N-terminal (aa 1–389) and C-terminal (aa 389–523) halves of DLIC. Bar graph shows the mean ratio of SEPT9 to GST or GST-DLIC protein band intensities from three independent experiments; error bars indicate SEM. Schematic depicts the GTPase-like and adaptor-binding domains of DLIC. (C) Coomassie-stained gel shows results from pull-down assays of His-SEPT9 with GST (control) or GST-tagged DIC(1–108), DIC(108–268), and DIC(1–268). Schematic shows the major domains of DIC and their corresponding interactions with components of the dynein–dynactin complex. Bar graph shows quantification of the relative amount of SEPT9 pulled down as the ratio (mean ± SEM) of SEPT9 to GST protein band intensities from five independent experiments. (D) Coomassie-stained gel (top) and Western blot (bottom; anti-His) show results from pull-down assays of recombinant NTE and G domain fragments of His-SEPT9 with GST-DIC(108–268). (E) Bar graph shows the mean ratio of NTE or G domain fragments of SEPT9 to GST-DIC(108–268) protein band intensities from three independent experiments; error bars indicate SEM. (F) Gel shows the input and results from protein binding assays of equimolar (0.5 µM) recombinant p150GLUED-CC1-Halo-His with GST-DIC(1–268) in the presence of increasing concentrations of His-mCherry-SEPT9 (0, 5, and 10 µM). Bar graph shows the relative increase in the amount of DIC(1–268)-bound p150GLUED-CC1 with increasing concentrations of SEPT9 from three independent experiments; ratio was set to 100 for 0 µM of SEPT9. Error bars indicate SEM.

SEPT9 provides a molecular link between dynein and dynactin through its GTP-binding and NTE domains, respectively. (A) Coomassie-stained gel shows the recombinant NTE and G domains of SEPT9, which were used in pull-down assays with purified dynein and dynactin. Pull-downs were Western blotted with antibodies against DHC and p150GLUED. Bar graph shows the mean ratio of DHC to NTE or G domain and p150GLUED to NTE or G domain fragments of His-SEPT9 intensities from three independent experiments; error bars indicate SEM. Schematic depicts the domains of SEPT9 and the position of the catalytic T339 residue required for GTPase activity. (B) Coomassie-stained gel shows the results of protein binding assays between recombinant His-SEPT9 and GST (control) or the GST-tagged N-terminal (aa 1–389) and C-terminal (aa 389–523) halves of DLIC. Bar graph shows the mean ratio of SEPT9 to GST or GST-DLIC protein band intensities from three independent experiments; error bars indicate SEM. Schematic depicts the GTPase-like and adaptor-binding domains of DLIC. (C) Coomassie-stained gel shows results from pull-down assays of His-SEPT9 with GST (control) or GST-tagged DIC(1–108), DIC(108–268), and DIC(1–268). Schematic shows the major domains of DIC and their corresponding interactions with components of the dynein–dynactin complex. Bar graph shows quantification of the relative amount of SEPT9 pulled down as the ratio (mean ± SEM) of SEPT9 to GST protein band intensities from five independent experiments. (D) Coomassie-stained gel (top) and Western blot (bottom; anti-His) show results from pull-down assays of recombinant NTE and G domain fragments of His-SEPT9 with GST-DIC(108–268). (E) Bar graph shows the mean ratio of NTE or G domain fragments of SEPT9 to GST-DIC(108–268) protein band intensities from three independent experiments; error bars indicate SEM. (F) Gel shows the input and results from protein binding assays of equimolar (0.5 µM) recombinant p150GLUED-CC1-Halo-His with GST-DIC(1–268) in the presence of increasing concentrations of His-mCherry-SEPT9 (0, 5, and 10 µM). Bar graph shows the relative increase in the amount of DIC(1–268)-bound p150GLUED-CC1 with increasing concentrations of SEPT9 from three independent experiments; ratio was set to 100 for 0 µM of SEPT9. Error bars indicate SEM.

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