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Cytoskeleton Inc biotinylated tubulin
Biotinylated Tubulin, supplied by Cytoskeleton Inc, used in various techniques. Bioz Stars score: 96/100, based on 196 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/biotinylated+tubulin/Tubulin+protein+-+biotin+porcine+brain/bio_rxiv__64898__2026__05__14__725143-196-16-18
Average 96 stars, based on 196 article reviews

biotinylated tubulin - by Bioz Stars, 2026-09

96/100 stars

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Residue:

Article Title: The P3018S disease variant reveals how dynein’s trailing motor sets ensemble velocity
Article Snippet: Two microliters of 10 mg/mL tubulin (Cytoskeleton, #T240-B) were mixed with 2 μL of 1 mg/mL biotinylated tubulin (Cytoskeleton, #T333P-A) and 1 μL of 10 mM GTP.. The mixture was incubated at 37 °C for 20 minutes.The mixture was incubated at 37 °C for 20 minutes.

Article Title: Adaptor-mediated recruitment of three dyneins to dynactin enhances force generation
Article Snippet: Two microlitres of 10 mg ml −1 tubulin (Cytoskeleton, T240-B) were mixed with 2 μl of 1 mg ml −1 biotinylated tubulin (Cytoskeleton, T333P-A) and 1 μl of 10 mM GTP.. The mixture was incubated at 37 °C for 20 min. After incubation, 0.5 μl of 0.2 mM paclitaxel in dimethylsulfoxide was added, and the incubation was continued for an additional 20 min.The mixture was incubated at 37 °C for 20 min. After incubation, 0.5 μl of 0.2 mM paclitaxel in dimethylsulfoxide was added, and the incubation was continued for an additional 20 min.

Article Title: Distinct Clinical Phenotypes in KIF1A-Associated Neurological Disorders Result from Different Amino Acid Substitutions at the Same Residue in KIF1A.
Article Snippet: To polymerize microtubules, 2 µL of 10 mg/mL tubulin (#T240-B, Cytoskeleton Inc., Denver, CO, USA) was mixed with 2 µL of 1 mg/mL biotinylated tubulin (#T333P-A, Cytoskeleton Inc.), 1 µL of 1 mg/mL HiLyte488-labeled tubulin (#TL488M-A, Cytoskeleton Inc.), and 1 µL of 10 mM GTP. .. T

Article Title: Distinct Clinical Phenotypes in KIF1A-Associated Neurological Disorders Result from Different Amino Acid Substitutions at the Same Residue in KIF1A
Article Snippet: To polymerize microtubules, 2 µL of 10 mg/mL tubulin (#T240-B, Cytoskeleton Inc., Denver, CO, USA) was mixed with 2 µL of 1 mg/mL biotinylated tubulin (#T333P-A, Cytoskeleton Inc.), 1 µL of 1 mg/mL HiLyte488-labeled tubulin (#TL488M-A, Cytoskeleton Inc.), and 1 µL of 10 mM GTP. .. T

Article Title: Distinct Clinical Phenotypes in KIF1A-Associated Neurological Disorders Result from Different Amino Acid Substitutions at the Same Residue in KIF1A
Article Snippet: To polymerize microtubules, 2 µL of 10 mg/mL tubulin (Cytoskeleton, #T240-B) was mixed with 2 µL of 1 mg/mL biotinylated tubulin (Cytoskeleton, #T333P-A), 1 µL of 1 mg/mL HiLyte488-labeled tubulin (Cytoskeleton, #TL488M-A), and 1 µL of 10 mM GTP. .. T

Article Title: A Two-Heads-Bound State Drives KIF1A Superprocessivity
Article Snippet: To prepare stabilized MTs, 2 µL of 10 mg/mL tubulin (Cytoskeleton, #T240-B) was mixed with 2 µL of 1 mg/mL biotinylated tubulin (Cytoskeleton, #T333P-A), 1 µl of 1 mg/ml HiLyte488-labeled tubulin (Cytoskeleton, #TL488M-A), and 1 µL of 10 mM GTP. .. T

Article Title: The Power of Three: Dynactin associates with three dyneins under load for greater force production
Article Snippet: 2 µL of 10 mg/mL tubulin (Cytoskeleton, #T240-B) was mixed with 2 µL of 1 mg/mL biotinylated tubulin (Cytoskeleton, # T333P-A) and 1 µL of 10 mM GTP. .. T

Image Search Results

Representative movie from MD simulations showing interactions of the Y-αCTT with the tubulin body over 240 ns. The tubulin body is shown in cartoon and colored gray. The αCTT is shown in stick and colored yellow with the glutamate side chains in red. Residues in site 1 (green), site 2 (cyan), and site 3 (magenta) appear as spheres when the αCTT is forming salt bridges with those residues.

Journal: eLife

Article Title: Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation

doi: 10.7554/eLife.109308

Figure Lengend Snippet: Representative movie from MD simulations showing interactions of the Y-αCTT with the tubulin body over 240 ns. The tubulin body is shown in cartoon and colored gray. The αCTT is shown in stick and colored yellow with the glutamate side chains in red. Residues in site 1 (green), site 2 (cyan), and site 3 (magenta) appear as spheres when the αCTT is forming salt bridges with those residues.

Article Snippet: Peptide, recombinant protein , Biotinylated porcine brain tubulin , Cytoskeleton , Cat# T333P , .

Techniques:

( A, B ) Schematic of tubulin protein and its conformational states within the microtubule lattice. ( A ) Schematic of tubulin heterodimer with the unstructured C-terminal tails (CTTs) protruding from the body of α- and β-tubulin. ( B ) Tubulin adds to the end of a microtubule in a GTP-bound and expanded state, resulting in a stabilizing GTP cap. In the microtubule lattice, β-tubulin undergoes GTP hydrolysis, resulting in a GDP lattice and compaction of α-tubulin. ( C ) Schematic of three sensors (YL1/2, A1aY1, and 4xCAPGly) generated to detect the accessibility of the Y-αCTT along the microtubule lattice. ( D, E ) Generation and validation of the YL1/2 Fab probe. ( D ) Schematic of antibody proteins. YL1/2 IgG: typical mammalian IgG molecule containing two heavy (H) and two light (L) chains. Light chains are comprised of one variable (V L , light orange) and one constant (C L , dark orange) region. Heavy chains are comprised of one variable (V H , light gray) and three constant (C H1–3 , dark gray) regions. Red dots indicate complementarity determining regions (CDRs) and purple lines indicate disulfide bonds. rMAb-EGFP: recombinant monoclonal antibody (rMAb) with EGFP (green star) fused to the C-terminus of the light chain. YL1/2 Fab -EGFP: Fragment antibody binding (Fab) produced from rMAb-EGFP by papain cleavage. ( E ) GST-tagged αCTT sequences were probed by western blotting with (left) commercial YL1/2 monoclonal antibody, (middle) rMAb-YL1/2-EGFP, or (right) YL1/2 Fab -EGFP. Each blot was also probed for GST protein as a loading control. Y: full-length and tyrosinated αCTT sequence; ΔY: detyrosinated αCTT sequence (lacking the C-terminal tyrosine); ΔC2: αCTT sequence lacking the C-terminal two amino acids. ( F ) Schematic of synthetic protein A1aY1 tagged at its N-terminus with sTag-RFP (red star). ( G ) Schematic of the domain organization of (top) full-length CLIP-170 and (bottom) the 4xCAPGly probe tagged at its C-terminus with mEGFP (green star). ( H, I ) Representative images of ( H ) YL1/2 Fab -EGFP or ( I ) 4xCAPGly-mEGFP proteins binding to tyrosinated (Y–MT) or detyrosinated (ΔY-MT) microtubules. HeLa tubulin was polymerized into microtubules and Taxol-stabilized. The microtubules were used directly (Y-MTs) or detyrosinated by VASH/SVBP-containing lysate before adding the ( H ) YL1/2 Fab -EGFP or ( I ) 4xCAPGly-mEGFP probes. Scale bars: 5 µm. Figure 1—source data 1. TIFF files of original western blots. Figure 1—source data 2. PDF file containing original gels for panel E indicating the relevant bands.

Journal: eLife

Article Title: Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation

doi: 10.7554/eLife.109308

Figure Lengend Snippet: ( A, B ) Schematic of tubulin protein and its conformational states within the microtubule lattice. ( A ) Schematic of tubulin heterodimer with the unstructured C-terminal tails (CTTs) protruding from the body of α- and β-tubulin. ( B ) Tubulin adds to the end of a microtubule in a GTP-bound and expanded state, resulting in a stabilizing GTP cap. In the microtubule lattice, β-tubulin undergoes GTP hydrolysis, resulting in a GDP lattice and compaction of α-tubulin. ( C ) Schematic of three sensors (YL1/2, A1aY1, and 4xCAPGly) generated to detect the accessibility of the Y-αCTT along the microtubule lattice. ( D, E ) Generation and validation of the YL1/2 Fab probe. ( D ) Schematic of antibody proteins. YL1/2 IgG: typical mammalian IgG molecule containing two heavy (H) and two light (L) chains. Light chains are comprised of one variable (V L , light orange) and one constant (C L , dark orange) region. Heavy chains are comprised of one variable (V H , light gray) and three constant (C H1–3 , dark gray) regions. Red dots indicate complementarity determining regions (CDRs) and purple lines indicate disulfide bonds. rMAb-EGFP: recombinant monoclonal antibody (rMAb) with EGFP (green star) fused to the C-terminus of the light chain. YL1/2 Fab -EGFP: Fragment antibody binding (Fab) produced from rMAb-EGFP by papain cleavage. ( E ) GST-tagged αCTT sequences were probed by western blotting with (left) commercial YL1/2 monoclonal antibody, (middle) rMAb-YL1/2-EGFP, or (right) YL1/2 Fab -EGFP. Each blot was also probed for GST protein as a loading control. Y: full-length and tyrosinated αCTT sequence; ΔY: detyrosinated αCTT sequence (lacking the C-terminal tyrosine); ΔC2: αCTT sequence lacking the C-terminal two amino acids. ( F ) Schematic of synthetic protein A1aY1 tagged at its N-terminus with sTag-RFP (red star). ( G ) Schematic of the domain organization of (top) full-length CLIP-170 and (bottom) the 4xCAPGly probe tagged at its C-terminus with mEGFP (green star). ( H, I ) Representative images of ( H ) YL1/2 Fab -EGFP or ( I ) 4xCAPGly-mEGFP proteins binding to tyrosinated (Y–MT) or detyrosinated (ΔY-MT) microtubules. HeLa tubulin was polymerized into microtubules and Taxol-stabilized. The microtubules were used directly (Y-MTs) or detyrosinated by VASH/SVBP-containing lysate before adding the ( H ) YL1/2 Fab -EGFP or ( I ) 4xCAPGly-mEGFP probes. Scale bars: 5 µm. Figure 1—source data 1. TIFF files of original western blots. Figure 1—source data 2. PDF file containing original gels for panel E indicating the relevant bands.

Article Snippet: Peptide, recombinant protein , Biotinylated porcine brain tubulin , Cytoskeleton , Cat# T333P , .

Techniques: Generated, Biomarker Discovery, Recombinant, Binding Assay, Produced, Western Blot, Control, Sequencing

COS-7 cells transiently expressing sTagRFP-A1aY1 and mEGFP were ( A ) imaged live, ( B ) fixed and mounted, or ( C ) fixed and stained for total tubulin (microtubules). Magnified views of the red and purple boxed regions are shown to the right. Scale bars: 10 µm for whole-cell views and for magnified views.

Journal: eLife

Article Title: Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation

doi: 10.7554/eLife.109308

Figure Lengend Snippet: COS-7 cells transiently expressing sTagRFP-A1aY1 and mEGFP were ( A ) imaged live, ( B ) fixed and mounted, or ( C ) fixed and stained for total tubulin (microtubules). Magnified views of the red and purple boxed regions are shown to the right. Scale bars: 10 µm for whole-cell views and for magnified views.

Article Snippet: Peptide, recombinant protein , Biotinylated porcine brain tubulin , Cytoskeleton , Cat# T333P , .

Techniques: Expressing, Staining

COS-7 cells transiently expressing 4xCAPGly-mSc3 and mEGFP were ( A ) imaged live, ( B ) fixed and mounted, or ( C ) fixed and stained for total tubulin (microtubules). Magnified views of the red and purple boxed regions are shown to the right. Scale bars: 10 µm for whole-cell views and for magnified views.

Journal: eLife

Article Title: Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation

doi: 10.7554/eLife.109308

Figure Lengend Snippet: COS-7 cells transiently expressing 4xCAPGly-mSc3 and mEGFP were ( A ) imaged live, ( B ) fixed and mounted, or ( C ) fixed and stained for total tubulin (microtubules). Magnified views of the red and purple boxed regions are shown to the right. Scale bars: 10 µm for whole-cell views and for magnified views.

Article Snippet: Peptide, recombinant protein , Biotinylated porcine brain tubulin , Cytoskeleton , Cat# T333P , .

Techniques: Expressing, Staining

( A, B ) Representative live-cell images of ( A ) A1aY1 or ( B ) 4xCAPGly HeLa stable cell lines transiently expressing the indicated mEGFP-tagged tubulin or MAP constructs. Cell boundaries are indicated by blue dotted lines. Scale bars: 20 µm. ( C, D ) Quantification of ( C ) sTag-RFP-A1aY1 or ( D ) CAPGly-mSc3 probe colocalization with mEGFP-tagged tubulin or MAP constructs. The threshold overlap score (TOS) was measured on a per-cell basis where 1.0 indicates perfect colocalization, –1.0 indicates perfect anti-colocalization, and values near 0 indicate no relationship. Data from three independent experiments are presented as Tukey box plots. The box encompasses the 25th to 75th percentiles, with a line at the median. Whiskers show the last data point within 1.5 times the interquartile range. Outliers are plotted as individual points. *: p<0.1; **: p<0.001; ****: p<0.0001; ns: not significant (Kruskal-Wallis test followed by post-hoc Dunn’s multiple pairwise comparisons with TubA1A as the control). Number of cells analyzed (n) in ( C ): TubA1A=69, Tau = 57, MAP2=66, Kif5Cr igor = 70, CAMSAP2=50, CAMSAP3=56, and MAP7=55 and in ( D ): TubA1A=62, Tau = 61, MAP2=61, Kif5C rigor = 76, CAMSAP2=62, CAMSAP3=71, and MAP7=57. ( E ) Schematic model depicting how expander and compactor MAPs regulate microtubule lattice conformation, influencing Y-αCTT accessibility. Figure 3—source data 1. Excel file with fluorescence intensity measurements.

Journal: eLife

Article Title: Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation

doi: 10.7554/eLife.109308

Figure Lengend Snippet: ( A, B ) Representative live-cell images of ( A ) A1aY1 or ( B ) 4xCAPGly HeLa stable cell lines transiently expressing the indicated mEGFP-tagged tubulin or MAP constructs. Cell boundaries are indicated by blue dotted lines. Scale bars: 20 µm. ( C, D ) Quantification of ( C ) sTag-RFP-A1aY1 or ( D ) CAPGly-mSc3 probe colocalization with mEGFP-tagged tubulin or MAP constructs. The threshold overlap score (TOS) was measured on a per-cell basis where 1.0 indicates perfect colocalization, –1.0 indicates perfect anti-colocalization, and values near 0 indicate no relationship. Data from three independent experiments are presented as Tukey box plots. The box encompasses the 25th to 75th percentiles, with a line at the median. Whiskers show the last data point within 1.5 times the interquartile range. Outliers are plotted as individual points. *: p<0.1; **: p<0.001; ****: p<0.0001; ns: not significant (Kruskal-Wallis test followed by post-hoc Dunn’s multiple pairwise comparisons with TubA1A as the control). Number of cells analyzed (n) in ( C ): TubA1A=69, Tau = 57, MAP2=66, Kif5Cr igor = 70, CAMSAP2=50, CAMSAP3=56, and MAP7=55 and in ( D ): TubA1A=62, Tau = 61, MAP2=61, Kif5C rigor = 76, CAMSAP2=62, CAMSAP3=71, and MAP7=57. ( E ) Schematic model depicting how expander and compactor MAPs regulate microtubule lattice conformation, influencing Y-αCTT accessibility. Figure 3—source data 1. Excel file with fluorescence intensity measurements.

Article Snippet: Peptide, recombinant protein , Biotinylated porcine brain tubulin , Cytoskeleton , Cat# T333P , .

Techniques: Stable Transfection, Expressing, Construct, Control, Fluorescence

( A ) Representative images of HeLa cells transiently expressing the indicated mEGFP-tagged MAPs and then fixed and stained with antibodies against detyrosinated microtubules (∆Y-tubulin) and total microtubules (MTs). Images are shown in inverted grayscale. The nuclei are represented by blue pseudocolor in the bottom panels. Blue dotted lines: boundaries of cells expressing the corresponding MAPs. Scale bars: 20 µm.( B ) Quantification of the intensity of detyrosination on MAP-bound microtubules. The fluorescence intensity of detyrosination was measured on MAP-decorated microtubules and normalized against the total microtubule intensity of MAP-decorated microtubules. Data from three independent experiments are presented as Tukey box plots. ****: p<0.0001; ns: not significant (Kruskal-Wallis test followed by post-hoc Dunn’s multiple pairwise comparisons with tau as the control). Number of cells analyzed (n): Tau = 67, MAP2=70, Kif5Cr igor = 75, CAMSAP2=67, CAMSAP3=70, and MAP7=67. ( C ) Quantification of the colocalization of MAPs and detyrosinated microtubules. The threshold overlap score (TOS) was measured on a per-cell basis. Data from three independent experiments are presented as Tukey box plots. The box encompasses the 25th to 75th percentiles, with a line at the median. Whiskers show the last data point within 1.5 times the interquartile range. Outliers are plotted as individual points. ****: p<0.0001; ns: not significant (Kruskal-Wallis test followed by post-hoc Dunn’s multiple pairwise comparisons with tau as the control). Number of cells analyzed (n): Tau = 70, MAP2=72, Kif5Cr igor = 76, CAMSAP2=66, CAMSAP3=69, and MAP7=67. Figure 4—source data 1. Excel file with fluorescence intensity measurements.

Journal: eLife

Article Title: Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation

doi: 10.7554/eLife.109308

Figure Lengend Snippet: ( A ) Representative images of HeLa cells transiently expressing the indicated mEGFP-tagged MAPs and then fixed and stained with antibodies against detyrosinated microtubules (∆Y-tubulin) and total microtubules (MTs). Images are shown in inverted grayscale. The nuclei are represented by blue pseudocolor in the bottom panels. Blue dotted lines: boundaries of cells expressing the corresponding MAPs. Scale bars: 20 µm.( B ) Quantification of the intensity of detyrosination on MAP-bound microtubules. The fluorescence intensity of detyrosination was measured on MAP-decorated microtubules and normalized against the total microtubule intensity of MAP-decorated microtubules. Data from three independent experiments are presented as Tukey box plots. ****: p<0.0001; ns: not significant (Kruskal-Wallis test followed by post-hoc Dunn’s multiple pairwise comparisons with tau as the control). Number of cells analyzed (n): Tau = 67, MAP2=70, Kif5Cr igor = 75, CAMSAP2=67, CAMSAP3=70, and MAP7=67. ( C ) Quantification of the colocalization of MAPs and detyrosinated microtubules. The threshold overlap score (TOS) was measured on a per-cell basis. Data from three independent experiments are presented as Tukey box plots. The box encompasses the 25th to 75th percentiles, with a line at the median. Whiskers show the last data point within 1.5 times the interquartile range. Outliers are plotted as individual points. ****: p<0.0001; ns: not significant (Kruskal-Wallis test followed by post-hoc Dunn’s multiple pairwise comparisons with tau as the control). Number of cells analyzed (n): Tau = 70, MAP2=72, Kif5Cr igor = 76, CAMSAP2=66, CAMSAP3=69, and MAP7=67. Figure 4—source data 1. Excel file with fluorescence intensity measurements.

Article Snippet: Peptide, recombinant protein , Biotinylated porcine brain tubulin , Cytoskeleton , Cat# T333P , .

Techniques: Expressing, Staining, Fluorescence, Control

( A,B ) Western blot of HeLa cells transiently expressing internal PA-tagged α-tubulin TubA1A. ( A ) sTagRFP-A1aY1 stable HeLa cells or ( B ) 4xCAPGly-mSc3 stable HeLa cells were untransfected (untr.) or transfected with plasmids for expressing PA-tagged WT or E254A α-tubulin (TubA1A). Whole cell lysates were prepared and analyzed by immunoblotting with the antibodies indicated on the left side of the blots. Size markers in kD are indicated on the right side of the blots. Asterisks denote upshifted PA-tagged tubulin bands. Figure 5—figure supplement 1—source data 1. PDF file containing original gels for panels A and B indicating the relevant bands. Figure 5—figure supplement 1—source data 2. TIFF files of original western blots.

Journal: eLife

Article Title: Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation

doi: 10.7554/eLife.109308

Figure Lengend Snippet: ( A,B ) Western blot of HeLa cells transiently expressing internal PA-tagged α-tubulin TubA1A. ( A ) sTagRFP-A1aY1 stable HeLa cells or ( B ) 4xCAPGly-mSc3 stable HeLa cells were untransfected (untr.) or transfected with plasmids for expressing PA-tagged WT or E254A α-tubulin (TubA1A). Whole cell lysates were prepared and analyzed by immunoblotting with the antibodies indicated on the left side of the blots. Size markers in kD are indicated on the right side of the blots. Asterisks denote upshifted PA-tagged tubulin bands. Figure 5—figure supplement 1—source data 1. PDF file containing original gels for panels A and B indicating the relevant bands. Figure 5—figure supplement 1—source data 2. TIFF files of original western blots.

Article Snippet: Peptide, recombinant protein , Biotinylated porcine brain tubulin , Cytoskeleton , Cat# T333P , .

Techniques: Western Blot, Expressing, Transfection

( A–D ) Live-cell imaging of Y-αCTT probes. ( A, C ) Representative images of ( A ) sTagRFP-A1aY1 or ( C ) 4xCAPGly-mSc3 HeLa stable cell lines transiently expressing PA-tagged WT or E254A α-tubulin with an IRES-driven mEGFP protein as a reporter of transfected cells. Cell boundaries are indicated by blue dotted lines. Scale bars: 20 µm. ( B, D ) Quantification of ( B ) A1aY1 or ( D ) 4xCAPGly probe binding to microtubules. The density was measured as the ratio of the skeletonized probe-decorated microtubule length to the total cell area. Data from three independent experiments are presented as Tukey box plots. The box encompasses the 25th to 75th percentiles, with a line at the median. Whiskers show the last data point within 1.5 times the interquartile range. Outliers are plotted as individual points. **: p<0.01 (Mann-Whitney U test). Number of cells analyzed (n) in ( B ): WT = 33, E254A=29 and in ( D ): WT = 42, E254A=50. ( E,F ) Detyrosinated microtubules. ( E ) Representative images of HeLa cells transiently expressing PA-tagged WT or E254A α-tubulin (TubA1A) and then fixed and stained with antibodies against the PA tag, detyrosinated microtubules (∆Y-tubulin), and total microtubules (MTs). Images are shown in inverted grayscale. The nuclei are represented by blue pseudocolor in the bottom panels. Blue dotted lines: boundaries of cells expressing α-tubulin. Scale bars: 20 µm. ( F ) Quantification of the intensity of detyrosination in cells expressing PA-tagged WT or E254A α-tubulin. The fluorescence intensity of detyrosination was measured on a per-cell basis and normalized against the total microtubule intensity. Data from three independent experiments are presented as Tukey box plots. ****: p<0.0001; ns: not significant (Kruskal-Wallis test followed by post-hoc Dunn’s multiple pairwise comparisons with the untransfected sample (untr.) as the control). Number of cells analyzed (n): untransfected = 105, WT = 84, E254A=94. Figure 5—source data 1. Excel file with fluorescence intensity measurements.

Journal: eLife

Article Title: Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation

doi: 10.7554/eLife.109308

Figure Lengend Snippet: ( A–D ) Live-cell imaging of Y-αCTT probes. ( A, C ) Representative images of ( A ) sTagRFP-A1aY1 or ( C ) 4xCAPGly-mSc3 HeLa stable cell lines transiently expressing PA-tagged WT or E254A α-tubulin with an IRES-driven mEGFP protein as a reporter of transfected cells. Cell boundaries are indicated by blue dotted lines. Scale bars: 20 µm. ( B, D ) Quantification of ( B ) A1aY1 or ( D ) 4xCAPGly probe binding to microtubules. The density was measured as the ratio of the skeletonized probe-decorated microtubule length to the total cell area. Data from three independent experiments are presented as Tukey box plots. The box encompasses the 25th to 75th percentiles, with a line at the median. Whiskers show the last data point within 1.5 times the interquartile range. Outliers are plotted as individual points. **: p<0.01 (Mann-Whitney U test). Number of cells analyzed (n) in ( B ): WT = 33, E254A=29 and in ( D ): WT = 42, E254A=50. ( E,F ) Detyrosinated microtubules. ( E ) Representative images of HeLa cells transiently expressing PA-tagged WT or E254A α-tubulin (TubA1A) and then fixed and stained with antibodies against the PA tag, detyrosinated microtubules (∆Y-tubulin), and total microtubules (MTs). Images are shown in inverted grayscale. The nuclei are represented by blue pseudocolor in the bottom panels. Blue dotted lines: boundaries of cells expressing α-tubulin. Scale bars: 20 µm. ( F ) Quantification of the intensity of detyrosination in cells expressing PA-tagged WT or E254A α-tubulin. The fluorescence intensity of detyrosination was measured on a per-cell basis and normalized against the total microtubule intensity. Data from three independent experiments are presented as Tukey box plots. ****: p<0.0001; ns: not significant (Kruskal-Wallis test followed by post-hoc Dunn’s multiple pairwise comparisons with the untransfected sample (untr.) as the control). Number of cells analyzed (n): untransfected = 105, WT = 84, E254A=94. Figure 5—source data 1. Excel file with fluorescence intensity measurements.

Article Snippet: Peptide, recombinant protein , Biotinylated porcine brain tubulin , Cytoskeleton , Cat# T333P , .

Techniques: Live Cell Imaging, Stable Transfection, Expressing, Transfection, Binding Assay, MANN-WHITNEY, Staining, Fluorescence, Control

$( A ) Representative image from MD simulations identifying four distinct sites where the Y-αCTT interacts with the body of tubulin subunits in the microtubule: sites 1 (green) and 2 (cyan) are on the adjacent β-tubulin along a protofilament (i.e. next tubulin towards the microtubule minus end), whereas sites 3 (purple) and 4 (salmon) are cis-interactions with α-tubulin itself. The tubulin body is shown in cartoon and colored gray. The Y-αCTT is shown in stick and colored yellow with the aspartate and glutamate side chains in red: 438-DSVEGEGEEEGEEY-451. ( B ) Interaction rate of the glutamate residues in the Y-αCTT with the four tubulin body sites for microtubules in the (gold) GDP or (blue) GTP states. ( C ) The fraction of Y-αCTTs that are inaccessible as a function of time for microtubules in the (gold) GDP or (blue) GTP state where inaccessibility is defined as one or more salt bridges formed between glutamates E445-E450 and the interaction sites in the microtubule body.$

Journal: eLife

Article Title: Accessibility of the unstructured α-tubulin C-terminal tail is controlled by microtubule lattice conformation

doi: 10.7554/eLife.109308

Figure Lengend Snippet: ( A ) Representative image from MD simulations identifying four distinct sites where the Y-αCTT interacts with the body of tubulin subunits in the microtubule: sites 1 (green) and 2 (cyan) are on the adjacent β-tubulin along a protofilament (i.e. next tubulin towards the microtubule minus end), whereas sites 3 (purple) and 4 (salmon) are cis-interactions with α-tubulin itself. The tubulin body is shown in cartoon and colored gray. The Y-αCTT is shown in stick and colored yellow with the aspartate and glutamate side chains in red: 438-DSVEGEGEEEGEEY-451. ( B ) Interaction rate of the glutamate residues in the Y-αCTT with the four tubulin body sites for microtubules in the (gold) GDP or (blue) GTP states. ( C ) The fraction of Y-αCTTs that are inaccessible as a function of time for microtubules in the (gold) GDP or (blue) GTP state where inaccessibility is defined as one or more salt bridges formed between glutamates E445-E450 and the interaction sites in the microtubule body.

Article Snippet: Peptide, recombinant protein , Biotinylated porcine brain tubulin , Cytoskeleton , Cat# T333P , .

Techniques:

A. Representative kymographs showing GFP-UNC-45A and GFP-katanin binding to microtubules. 100nM GFP-UNC-45A or 25nM GFP-katanin were used for the binding assay. These kymographs were used to calculate the duration of association and binding frequency. B. Quantification of binding kinetics of GFP-UNC-45A and GFP-katanin to microtubules. N= 527 and 542 binding events were quantified for GFP-UNC-45A and GFP-katanin, respectively. C. Left , representative images of biotinylated MT (visualized with DIC) treated with 100µM taxol for 1 hr and repaired with 500nM rhodamine-tubulins (red) minus/plus 100nM GFP-UNC-45A (green). Right , quantification of fluorescent intensity of rhodamine-tubulins binding to MTs minus/plus UNC-45A and expressed as normalized free tubulin AFU. n= 100 MTs per condition were quantified. Error bars represent mean ± SD. D. Experimental set-up for microtubule-repair experiments in the presence of free tubulin and UNC-45A. E . Top four panels , representative images of biotinylated MT treated with 100µM taxol for 1 hr and exposed (or not, last four panels from the bottom-minus UNC-45A) to 250nM GFP-UNC-45A and increasing concentrations (250nM-5uM) of rhodamine-tubulin. Second four panels from top , green channel (UNC-45A). Third four panels from top, red channel (rhodamine tubulin). Fourth four panels from top, merged image of green and red channels. F. Quantification of fluorescent intensity of rhodamine-tubulins binding to microtubules expressed as AFU of tubulins bound to microtubules for different input of rhodamine tubulin. n= 80 MTs per condition from two independent experiments were quantified. Error bars represent mean ± SD. G . Mean ± SD of AFU of tubulins bound to microtubules is plotted over the input free tubulin. Error bars represent mean ± SD. H . Difference in mean rhodamine-tubulin AFU between minus/plus 250nM GFP-UNC-45A is plotted over the input free tubulin concentrations. n= 80 microtubules per condition were quantified. Error bars represent mean ± SE.

Journal: bioRxiv

Article Title: UNC-45A drives ATP-independent microtubule severing via defect recognition and repair inhibition, contributing to neurite dystrophy

doi: 10.1101/2025.08.26.671351

Figure Lengend Snippet: A. Representative kymographs showing GFP-UNC-45A and GFP-katanin binding to microtubules. 100nM GFP-UNC-45A or 25nM GFP-katanin were used for the binding assay. These kymographs were used to calculate the duration of association and binding frequency. B. Quantification of binding kinetics of GFP-UNC-45A and GFP-katanin to microtubules. N= 527 and 542 binding events were quantified for GFP-UNC-45A and GFP-katanin, respectively. C. Left , representative images of biotinylated MT (visualized with DIC) treated with 100µM taxol for 1 hr and repaired with 500nM rhodamine-tubulins (red) minus/plus 100nM GFP-UNC-45A (green). Right , quantification of fluorescent intensity of rhodamine-tubulins binding to MTs minus/plus UNC-45A and expressed as normalized free tubulin AFU. n= 100 MTs per condition were quantified. Error bars represent mean ± SD. D. Experimental set-up for microtubule-repair experiments in the presence of free tubulin and UNC-45A. E . Top four panels , representative images of biotinylated MT treated with 100µM taxol for 1 hr and exposed (or not, last four panels from the bottom-minus UNC-45A) to 250nM GFP-UNC-45A and increasing concentrations (250nM-5uM) of rhodamine-tubulin. Second four panels from top , green channel (UNC-45A). Third four panels from top, red channel (rhodamine tubulin). Fourth four panels from top, merged image of green and red channels. F. Quantification of fluorescent intensity of rhodamine-tubulins binding to microtubules expressed as AFU of tubulins bound to microtubules for different input of rhodamine tubulin. n= 80 MTs per condition from two independent experiments were quantified. Error bars represent mean ± SD. G . Mean ± SD of AFU of tubulins bound to microtubules is plotted over the input free tubulin. Error bars represent mean ± SD. H . Difference in mean rhodamine-tubulin AFU between minus/plus 250nM GFP-UNC-45A is plotted over the input free tubulin concentrations. n= 80 microtubules per condition were quantified. Error bars represent mean ± SE.

Article Snippet: Porcine brain tubulin (T240), rhodamine-labeled tubulin (TL590M), and biotinylated tubulin (T333) were purchased from Cytoskeleton.

Techniques: Binding Assay

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biotinylated tubulin (Cytoskeleton Inc)

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