Figure S2. Ate1 −/− cells do not show... | Rockefeller University Press

Figure S2.

$Ate1−/−cells do not show gross defects in the microtubule cytoskeleton. (A) Western blots for ATE1 (top, red), R-actin (middle, red), and GFP (bottom, green) in Ate1+/+ or Ate1−/− cells transfected with the arginylation sensor DD-β15-GFP and/or Ate1-GFP. (B) Schematic for the arginylation sensor used in A. Ub-DD-β15-GFP is expressed, and the ubiquitin is immediately cleaved, producing DD-β15-GFP. ATE1 can arginylate the N-terminal D, producing RDD-β15-GFP. (β15 is the first 15 amino acids of β-actin after N-terminal processing, DDIAALVVDNGSGMC.) (C) Images of Ate1−/− cells co-transfected with mch-EB3 (left) and Ate1-GFP (right). Scale bar = 10 μm. (D) Quantification of the EB3 track duration. Mean ± SEM, N = 4 (n = 20 cells per condition), one-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. Symbol colors indicate paired average measurements. Violin plot represents the distribution of average EB3 track duration for individual cells. (E) Western blot for ⍺-tubulin in Ate1+/+ or Ate1−/− cells. (F) Quantification of ⍺-tubulin normalized to total protein. Mean ± SD, N = 3, paired t test. Symbol colors indicate paired average measurements. (G) Immunofluorescence images of Ate1+/+ or Ate1−/− cells stained for ⍺-tubulin (green), γ-tubulin (magenta), and DAPI (blue). Scale bar = 20 μm. (H) Quantification of ⍺-tubulin per cell area. Mean ± SEM, N = 3 (n = 182–225 cells per condition), paired t test. Symbol colors indicate paired average measurements. Violin plot represents the distribution of ⍺-tubulin per cell area for individual cells. (I) Western blots for ⍺-tubulin on soluble and polymerized tubulin fractions from Ate1+/+ or Ate1−/− cells. (J) Quantification of the fraction of ⍺-tubulin in the polymerized tubulin fraction. Mean ± SEM, N = 3 (n = 12 total repeats per condition), Ate1+/+ versus Ate1−/−: ***P = 0.0005, paired t test. Symbol colors indicate paired average measurements. Truncated violin plot represents the fraction of polymerized tubulin for individual repeats. (K) Western blots for other tubulin post-translational modifications in Ate1+/+ or Ate1−/− cells: acetylation, detyrosination, tyrosination, glutamylation, and polyglutamylation. (L) Quantification of other tubulin post-translational modifications normalized to total protein. Mean ± SD, N = 3, paired t test. Symbol colors indicate paired average measurements. Source data are available for this figure: SourceData FS2. Refer to the image caption for details.$

Ate1 ^−/− cells do not show gross defects in the microtubule cytoskeleton. (A) Western blots for ATE1 (top, red), R-actin (middle, red), and GFP (bottom, green) in Ate1^+/+ or Ate1^−/− cells transfected with the arginylation sensor DD-β15-GFP and/or Ate1-GFP. (B) Schematic for the arginylation sensor used in A. Ub-DD-β15-GFP is expressed, and the ubiquitin is immediately cleaved, producing DD-β15-GFP. ATE1 can arginylate the N-terminal D, producing RDD-β15-GFP. (β15 is the first 15 amino acids of β-actin after N-terminal processing, DDIAALVVDNGSGMC.) (C) Images of Ate1^−/− cells co-transfected with mch-EB3 (left) and Ate1-GFP (right). Scale bar = 10 μm. (D) Quantification of the EB3 track duration. Mean ± SEM, N = 4 (n = 20 cells per condition), one-way RM ANOVA corrected for multiple comparisons. Symbol colors indicate paired average measurements. Symbol colors indicate paired average measurements. Violin plot represents the distribution of average EB3 track duration for individual cells. (E) Western blot for ⍺-tubulin in Ate1^+/+ or Ate1^−/− cells. (F) Quantification of ⍺-tubulin normalized to total protein. Mean ± SD, N = 3, paired t test. Symbol colors indicate paired average measurements. (G) Immunofluorescence images of Ate1^+/+ or Ate1^−/− cells stained for ⍺-tubulin (green), γ-tubulin (magenta), and DAPI (blue). Scale bar = 20 μm. (H) Quantification of ⍺-tubulin per cell area. Mean ± SEM, N = 3 (n = 182–225 cells per condition), paired t test. Symbol colors indicate paired average measurements. Violin plot represents the distribution of ⍺-tubulin per cell area for individual cells. (I) Western blots for ⍺-tubulin on soluble and polymerized tubulin fractions from Ate1^+/+ or Ate1^−/− cells. (J) Quantification of the fraction of ⍺-tubulin in the polymerized tubulin fraction. Mean ± SEM, N = 3 (n = 12 total repeats per condition), Ate1^+/+ versus Ate1^−/−: ***P = 0.0005, paired t test. Symbol colors indicate paired average measurements. Truncated violin plot represents the fraction of polymerized tubulin for individual repeats. (K) Western blots for other tubulin post-translational modifications in Ate1^+/+ or Ate1^−/− cells: acetylation, detyrosination, tyrosination, glutamylation, and polyglutamylation. (L) Quantification of other tubulin post-translational modifications normalized to total protein. Mean ± SD, N = 3, paired t test. Symbol colors indicate paired average measurements. Source data are available for this figure: SourceData FS2.