Figure 5.
Tub1 and Tub3 isotypes are balanced to set α-tubulin protein levels. (A) Schematic of gene composition for the GFP-Tub3 induction strain and time course of induction experiments. (B) Example images of GFP-Tub3 in cells at indicated induction time. Arrowheads indicate tubulin assemblies. Scale bar = 1 µm. Images are a maximum intensity projection of z series. (C) Quantification of whole cell GFP-Tub3 fluorescence intensity. Each dot represents a single cell and are colored by replicate, triangles represent the mean of each experiment. Bars are mean ± 95% CI. (D) Quantification of GFP-Tub3 fluorescence intensity in astral microtubules. Each dot represents a single microtubule in a single cell and are colored by replicate, triangles represent the mean of each experiment. P value between wild type and mutant based on t test after one-way ANOVA. Bars are mean ± 95% CI. (E) Representative Western blot of GFP-Tub3 induction. Blots were probed for α- or β-tubulin, and Zwf1 (G6PD) as a loading control. (F) Quantification of total α- and β-tubulin levels across GFP-Tub3 induction. Dots are mean ± SD from three independent experiments, normalized to the level of α- or β-tubulin prior to induction. (G) Quantification of Tub1 and GFP-Tub3 levels across induction. Dots are mean ± SD from three independent experiments, normalized to the Tub1 levels prior to induction. P values were determined by t test after a one-way ANOVA. (H) Representative Western blot of tubulin levels after GFP-Tub3 shut off. Blots were probed for α- or β-tubulin, and Zwf1 (G6PD) as a loading control. (I) Quantification of total α- and β-tubulin levels after GFP-Tub3 shut off. Dots are mean ± SD from four independent experiments, normalized to the α- or β-tubulin levels at shutoff (t = 0). P values were determined by t test compared to t = 0, after a one-way ANOVA. Only P values <0.05 are shown. (J) Quantification of Tub1 and GFP-Tub3 levels after GFP-Tub3 shut off. Dots are mean ± SD from four independent experiments, normalized to the Tub1 or GFP-Tub3 levels at shutoff (t = 0). P values were determined by t test compared to t = 0, after a one-way ANOVA. Only P values <0.05 are shown. Dashed line represents the estimated loss of protein due to titration by cell division, calculated from the increase in Zwf1 signal. (K) Representative Western blot of Tub2-6xHis induction. Blots were probed for α- or β-tubulin, and Zwf1 (G6PD) as a loading control. (L) Quantification of total α- and β-tubulin levels across Tub2-6xHis induction. Dots are mean ± SD from five independent experiments, normalized to α- or β-tubulin levels prior to induction. (M) Quantification of Tub2-438Δ and Tub2-6xHis levels across Tub2-6xHis induction. Dots are mean ± SD from five independent experiments, normalized to Tub2-438Δ levels prior to induction. (N) Representative Western blot of Tub2-6xHis shut off. Blots were probed for α- or β-tubulin, and Zwf1 (G6PD) as a loading control. (O) Quantification of total α- and β-tubulin levels after Tub2-6xHis shut off. Dots are mean ± SD from four independent experiments, normalized to the α- or β-tubulin levels at shutoff (t = 0). (P) Quantification of Tub2-438Δ and Tub2-6xHis levels after Tub2-6xHis shut off. Dots are mean ± SD from four independent experiments, normalized to Tub2-438Δ or Tub2-6xHis levels at shutoff (t = 0). (Q) Representative Western blot of GFP-Tub3 induction with or without cycloheximide (CHX) treatment. (R) Quantification of Tub1 protein levels in indicated treatment groups. Data represent four experiments and are colored by day. Bars are mean ±95% CI. Source data are available for this figure: SourceData F5.

Tub1 and Tub3 isotypes are balanced to set α-tubulin protein levels. (A) Schematic of gene composition for the GFP-Tub3 induction strain and time course of induction experiments. (B) Example images of GFP-Tub3 in cells at indicated induction time. Arrowheads indicate tubulin assemblies. Scale bar = 1 µm. Images are a maximum intensity projection of z series. (C) Quantification of whole cell GFP-Tub3 fluorescence intensity. Each dot represents a single cell and are colored by replicate, triangles represent the mean of each experiment. Bars are mean ± 95% CI. (D) Quantification of GFP-Tub3 fluorescence intensity in astral microtubules. Each dot represents a single microtubule in a single cell and are colored by replicate, triangles represent the mean of each experiment. P value between wild type and mutant based on t test after one-way ANOVA. Bars are mean ± 95% CI. (E) Representative Western blot of GFP-Tub3 induction. Blots were probed for α- or β-tubulin, and Zwf1 (G6PD) as a loading control. (F) Quantification of total α- and β-tubulin levels across GFP-Tub3 induction. Dots are mean ± SD from three independent experiments, normalized to the level of α- or β-tubulin prior to induction. (G) Quantification of Tub1 and GFP-Tub3 levels across induction. Dots are mean ± SD from three independent experiments, normalized to the Tub1 levels prior to induction. P values were determined by t test after a one-way ANOVA. (H) Representative Western blot of tubulin levels after GFP-Tub3 shut off. Blots were probed for α- or β-tubulin, and Zwf1 (G6PD) as a loading control. (I) Quantification of total α- and β-tubulin levels after GFP-Tub3 shut off. Dots are mean ± SD from four independent experiments, normalized to the α- or β-tubulin levels at shutoff (t = 0). P values were determined by t test compared to t = 0, after a one-way ANOVA. Only P values <0.05 are shown. (J) Quantification of Tub1 and GFP-Tub3 levels after GFP-Tub3 shut off. Dots are mean ± SD from four independent experiments, normalized to the Tub1 or GFP-Tub3 levels at shutoff (t = 0). P values were determined by t test compared to t = 0, after a one-way ANOVA. Only P values <0.05 are shown. Dashed line represents the estimated loss of protein due to titration by cell division, calculated from the increase in Zwf1 signal. (K) Representative Western blot of Tub2-6xHis induction. Blots were probed for α- or β-tubulin, and Zwf1 (G6PD) as a loading control. (L) Quantification of total α- and β-tubulin levels across Tub2-6xHis induction. Dots are mean ± SD from five independent experiments, normalized to α- or β-tubulin levels prior to induction. (M) Quantification of Tub2-438Δ and Tub2-6xHis levels across Tub2-6xHis induction. Dots are mean ± SD from five independent experiments, normalized to Tub2-438Δ levels prior to induction. (N) Representative Western blot of Tub2-6xHis shut off. Blots were probed for α- or β-tubulin, and Zwf1 (G6PD) as a loading control. (O) Quantification of total α- and β-tubulin levels after Tub2-6xHis shut off. Dots are mean ± SD from four independent experiments, normalized to the α- or β-tubulin levels at shutoff (t = 0). (P) Quantification of Tub2-438Δ and Tub2-6xHis levels after Tub2-6xHis shut off. Dots are mean ± SD from four independent experiments, normalized to Tub2-438Δ or Tub2-6xHis levels at shutoff (t = 0). (Q) Representative Western blot of GFP-Tub3 induction with or without cycloheximide (CHX) treatment. (R) Quantification of Tub1 protein levels in indicated treatment groups. Data represent four experiments and are colored by day. Bars are mean ±95% CI. Source data are available for this figure: SourceData F5.

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