Figure 3. Microtubule nucleation induced by γ-TuNA in transfected cells. (A and B) Microtubule regrowth was performed on U2OS cells expressing 51–100 or its F75A mutant. (bottom) Enlarged views of the boxed areas are shown. The microtubule intensities of regrowth for 1 min were quantified from cells expressing the proteins at similar levels and were expressed relative to untransfected cells (B). At least 20 cells were measured in each of three independent experiments. (B) Error bars indicate mean ± SD. (C) Microtubule regrowth (1 min) was performed after γ-tubulin was depleted by RNAi. (left) The expressions of γ-tubulin and actin were examined by immunoblotting. (right) Cells transfected with 51–100 were subjected to the regrowth assay. The depletion of γ-tubulin reduced microtubule regrowth to 9.20 ± 2.89% of control cells (n = 60 cells for each quantification). (D) 51–100 (GFP tagged) was expressed in the background of endogenous CDK5RAP2 depleted by RNAi or in the control. (left) Cell extracts were immunoblotted for GFP-51–100 (anti-GFP), endogenous CDK5RAP2, and β-tubulin. (right) The cells were stained for microtubules (anti–α-tubulin) and endogenous CDK5RAP2 after microtubule regrowth for 1 min. Arrows denote centrosomes. (right) Enlarged views of the boxed areas are shown. Bars, 10 µm.
Figure 3.

Microtubule nucleation induced by γ-TuNA in transfected cells. (A and B) Microtubule regrowth was performed on U2OS cells expressing 51–100 or its F75A mutant. (bottom) Enlarged views of the boxed areas are shown. The microtubule intensities of regrowth for 1 min were quantified from cells expressing the proteins at similar levels and were expressed relative to untransfected cells (B). At least 20 cells were measured in each of three independent experiments. (B) Error bars indicate mean ± SD. (C) Microtubule regrowth (1 min) was performed after γ-tubulin was depleted by RNAi. (left) The expressions of γ-tubulin and actin were examined by immunoblotting. (right) Cells transfected with 51–100 were subjected to the regrowth assay. The depletion of γ-tubulin reduced microtubule regrowth to 9.20 ± 2.89% of control cells (n = 60 cells for each quantification). (D) 51–100 (GFP tagged) was expressed in the background of endogenous CDK5RAP2 depleted by RNAi or in the control. (left) Cell extracts were immunoblotted for GFP-51–100 (anti-GFP), endogenous CDK5RAP2, and β-tubulin. (right) The cells were stained for microtubules (anti–α-tubulin) and endogenous CDK5RAP2 after microtubule regrowth for 1 min. Arrows denote centrosomes. (right) Enlarged views of the boxed areas are shown. Bars, 10 µm.

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