Figure 5. Depletion of GCP4 disrupts the association of γ-TuNA with γ-TuRC. (A) siRNA-transfected HEK293T extracts were immunoblotted for γ-tubulin and GCPs. (B) Cell extracts were subjected to sucrose gradient centrifugation. An aliquot of each gradient fraction was analyzed by immunoblotting. (C) His-Flag–CDK5RAP2 (51–200) was used in the pull down of γ-TuCs from siRNA-transfected extracts. The anti-Flag precipitates were analyzed by immunoblotting. Note that the protein band below GCP4 is a heat shock protein bound nonspecifically to anti-Flag beads. (D) Microtubule regrowth was performed on U2OS cells with the regrowth time of 1 min. GCP4 depletion reduced microtubule regrowth to 14.02 ± 3.84% of control cells (n = 60 cells for each quantification). Bar, 10 µm. (E) Model for the activation of γ-TuRC–mediated microtubule nucleation by γ-TuNA. After γ-TuRC assembly, γ-TuNA binds to the complex to stimulate its nucleation of microtubules.
Figure 5.

Depletion of GCP4 disrupts the association of γ-TuNA with γ-TuRC. (A) siRNA-transfected HEK293T extracts were immunoblotted for γ-tubulin and GCPs. (B) Cell extracts were subjected to sucrose gradient centrifugation. An aliquot of each gradient fraction was analyzed by immunoblotting. (C) His-Flag–CDK5RAP2 (51–200) was used in the pull down of γ-TuCs from siRNA-transfected extracts. The anti-Flag precipitates were analyzed by immunoblotting. Note that the protein band below GCP4 is a heat shock protein bound nonspecifically to anti-Flag beads. (D) Microtubule regrowth was performed on U2OS cells with the regrowth time of 1 min. GCP4 depletion reduced microtubule regrowth to 14.02 ± 3.84% of control cells (n = 60 cells for each quantification). Bar, 10 µm. (E) Model for the activation of γ-TuRC–mediated microtubule nucleation by γ-TuNA. After γ-TuRC assembly, γ-TuNA binds to the complex to stimulate its nucleation of microtubules.

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