anti α β tubulin sheep antibody  (Cytoskeleton Inc)

Journal: The Plant Cell

Article Title: The unconventional TPX2 family protein TPXL3 regulates α Aurora kinase function in spindle morphogenesis in Arabidopsis

doi: 10.1093/plcell/koaf065

Figure Lengend Snippet: TPXL3 is required for the localization of α Aurora and γ-tubulin in the mitotic apparatus. The merged images have DNA in blue and corresponding green and magenta signals combined. A) A functional GFP-AUR1 fusion protein decorates spindle microtubules with biases toward spindle poles, leaving prominent microtubule signal near the metaphase plate in the merged image. B) In the amiR-TPXL3 mutant cells at metaphase, the GFP-AUR1 signal becomes mostly diffuse in the cytosol and no longer concentrated along spindle microtubules. C) Quantitative assessment shows that spindle-associated GFP-AUR1 signal is greatly compromised and replaced by diffuse cytoplasmic signals as revealed by the ratio of spindle and cytoplasm GFP-AUR1 signals assayed in 20 cells. Data as individual points are presented in box-and-whisker plots, showing the interquartile range (box), the median (horizontal line), and minimum and maximum values (whiskers). D–G) Comparative localizations of TPXL3-GFP and γ-tubulin in mitotic cells. At prophase, TPXL3 is heavily associated with the nuclear envelope where γ-tubulin begins to accumulate (D) . Both TPXL3 and γ-tubulin are loaded on kinetochore fibers as demonstrated in the anaphase cell (E) . At telophase, TPXL3 and γ-tubulin remain at polar regions while weak TPXL3 signal is detected proximal to the chromatid mass, but γ-tubulin is abundantly detected in the central spindle region (F) . During cytokinesis when γ-tubulin is heavily associated with the phragmoplast, TPXL3 can only be detected near the re-forming daughter nuclei (G) . H, I) At metaphase, γ-tubulin localization on spindle microtubules in the control cell is replaced by mostly diffuse signal in the cytoplasm of an amiR- TPXL3 cell. J) Quantitative assessment shows that spindle-associated γ-tubulin signal is compromised and replaced by diffuse cytoplasmic signals as revealed by the ratio of spindle and cytoplasm γ-tubulin signals assayed in 45 cells. Data as individual points are presented in box-and-whisker plots, showing the interquartile range (box), the median (horizontal line), and minimum and maximum values (whiskers). Scale bars, 5 µ m.

Article Snippet: The primary antibodies used here include the rabbit anti-GFP polyclonal antibody (1:400, ThermoFisher, catalog # A-6455), DM1A mouse anti-α-tubulin monoclonal antibody (1:400, Sigma-Aldrich, catalog # T9026), sheep anti-tubulin polyclonal antibodies (1:400, Cytoskeleton, catalog # ATN02), G9 mouse anti-γ-tubulin monoclonal antibody (1:400, ), and C2 mouse anti-FLAG monoclonal antibody (1:400, Shanghai Genomics, catalog # GNI4310-FG).

Techniques: Functional Assay, Mutagenesis, Whisker Assay, Control

Journal: The Plant Cell

Article Title: The unconventional TPX2 family protein TPXL3 regulates α Aurora kinase function in spindle morphogenesis in Arabidopsis

doi: 10.1093/plcell/koaf065

Figure Lengend Snippet: Compromised spindle apparatus is associated with retarded plant growth. A) Spindle microtubule arrays in metaphase cells indicated by the aligned chromosomes at the equatorial plane from indicated transgenic lines. While the amiRNA- TPXL3 mutant cell produces a spindle with disorganized spindle poles, the expression of the full-length microRNA-resistant TPXL3 R restores the spindle morphology. So is the truncated protein lacking C-terminal domain (TPXL3 I−IV ). While the deletion of domains I (TPXL3 II−V ) or II (TPXL3 ΔII ) does not render a toxic effect on the amiRNA- TPXL3 spindles, the deletion of domain IV (TPXL3 ΔIV ) or III–V (TPXL3 I−II ) greatly enhanced the deformation of spindles that have kinetochore MT fibers arranged in parallel bundles. The micrographs represent metaphase cells of root tips of at least 20 seedlings in each of 2 independent transgenic events for every construct, which show consistent results observed in both transgenic lines. B) The mean solidity of spindles is reduced to various degrees in the mutants expressing TPXL3 derivatives missing corresponding domain(s) when compared to the control cells by using cells processed for anti-tubulin immunofluorescence. The statistical significance of the difference between the amiR- TPXL3 cells ( n = 14) or amiR- TPXL3 cells expressing microRNA-resistant forms of TPXL3 derivatives (I–V, n = 15; II–V, n = 14; ΔII, n = 13; ΔIV, n = 14; I–IV, n = 15; or I + II, n = 15) and the wild-type ( n = 15) control cells is assessed by the U tests that result in averages ± standard deviations of 0.952 ± 0.015, 0.919 ± 0.018, 0.955 ± 0.012, 0.929 ± 0.024, 0.915 ± 0.029, 0.898 ± 0.051, 0.946 ± 0.016, and 0.912 ± 0.021 with P values of 3.575e-05, 0.722, 0.009383, 5.319e-05, 0.0001201, 0.3468, 3.288e-06, respectively. Asterisks indicate significant differences. C) The mean aspect ratio of spindles formed in mutant cells expressing TPXL3 derivatives missing corresponding domain(s) is reduced to various severities when compared to the control cells. The statistical significance of the difference between the amiR- TPXL3 cells or amiR- TPXL3 cells expressing microRNA-resistant forms of TPXL3 derivatives (I–V, II–V, ΔII, ΔIV, I–IV, or I + II) and the wild-type control cells is assessed by the U tests that result in averages ± standard deviations of 1.344 ± 0.172, 1.168 ± 0.097, 1.571 ± 0.240, 1.136 ± 0.290, 1.162 ± 0.197, 0.743 ± 0.097, 1.459 ± 0.208, and 0.772 ± 0.085 with P values of 0.0005233, 0.01202, 0.006743, 0.007796, 4.985e-08, 0.2752, 2.579e-08, respectively. Asterisks indicate significant differences. D) Growth phenotypes associated with the expression of various TPXL3 derivatives. While the expression of microRNA-resistant TPXL3 R greatly suppresses the growth phenotype of the amiRNA- TPXL3 plant, the expression of TPXL3 II−V or TPXL3 ΔII does not significantly alter the growth of the mutant. However, the expression of TPXL3 ΔIV or TPXL3 I−II further enhances growth defects as indicated by severe inhibition of axial growth and production of inflorescence. Representative plants are randomly selected from 5 independent transgenic lines that have been chosen from ten or more exhibiting consistent phenotypic characteristics for each construct. Data as individual points are presented in box-and-whisker plots, showing the interquartile range (box), the median (horizontal line), and minimum and maximum values (whiskers). Scale bars, 5 µ m ( A , applicable to all micrographs included) and 2 cm ( B ).

Article Snippet: The primary antibodies used here include the rabbit anti-GFP polyclonal antibody (1:400, ThermoFisher, catalog # A-6455), DM1A mouse anti-α-tubulin monoclonal antibody (1:400, Sigma-Aldrich, catalog # T9026), sheep anti-tubulin polyclonal antibodies (1:400, Cytoskeleton, catalog # ATN02), G9 mouse anti-γ-tubulin monoclonal antibody (1:400, ), and C2 mouse anti-FLAG monoclonal antibody (1:400, Shanghai Genomics, catalog # GNI4310-FG).

Techniques: Transgenic Assay, Mutagenesis, Expressing, Construct, Control, Immunofluorescence, Inhibition, Whisker Assay

Journal: iScience

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocyte meiotic spindle building

doi: 10.1016/j.isci.2024.110451

Figure Lengend Snippet: AURKA is required at early pro-metaphase I and metaphase I for spindle building (A) Experimental design of WT oocytes treated with MLN during oocyte maturation. Red arrows indicate MLN addition time; blue arrows indicate fixation time. (B, F, J) Schematic of spindles at indicated stages, and the processes analyzed. (C, G, K) Representative confocal images of oocytes fixed at the indicated stages with and without MLN, immunostained with PCNT (gray), TACC3 (magenta), Tubulin (green) and DAPI (blue). Scale bars 10 μm. (D, H, L) Quantification of aMTOC parameters in (A), (G), (K) respectively. (E, I, M) Quantification of spindle and LISD parameters in (A), (G), (K) respectively. Black dots: DMSO-treated; gray dots: MLN- treated. See also Figure S1 which contains statistics.

Article Snippet: Sheep polyclonal Alpha Tubulin antibody , Cytoskeleton , Cat#ATN02; RRID: AB_10708807.

Techniques:

Journal: iScience

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocyte meiotic spindle building

doi: 10.1016/j.isci.2024.110451

Figure Lengend Snippet: AURKC is required at late pro-metaphase I for aMTOC separation (A) Experimental design of BC-KO oocytes treated with MLN; Red arrows indicate MLN addition time; blue arrows indicate fixation time. (B, F, J) Schematic of spindles at the indicated stages, and the processes analyzed. (C, G, K) Representative confocal images of oocytes fixed at the indicated stages with and without MLN, immunostained with PCNT (gray), TACC3 (magenta), Tubulin (green) and DAPI (blue). Scale bars 10 μm. (D, H, L) Quantification of aMTOC parameters in (A), (G), (K), respectively. (E, I, M) Quantification of spindle and LISD parameters in (A), (G), (K), respectively. Black dots: DMSO- treated; gray dots: MLN-treated. See also Figure S2 which contains statistics.

Article Snippet: Sheep polyclonal Alpha Tubulin antibody , Cytoskeleton , Cat#ATN02; RRID: AB_10708807.

Techniques:

Journal: iScience

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocyte meiotic spindle building

doi: 10.1016/j.isci.2024.110451

Figure Lengend Snippet: AURKA and AURKC specific functions at late pro-metaphase I in mouse oocytes (A) Live cell light-sheet imaging of WT and BC- MLN-treated KO oocytes during Early pro-metaphase I; aMTOC (gray), SiR-tubulin (green), DNA (magenta). Time is indicated in h:min; Scale bar 10 μm (B, D) Quantification of spindle volume over time in WT (B) and BC-KO (D), respectively. (C, E) Quantification of area and aMTOC numbers over time in WT (C) and BC-KO (E), respectively. Yellow shadow: DMSO-treated; Purple shadow: MLN-treated. The red arrows indicate MLN addition time. Number of oocytes: WT DMSO: 11; WT MLN: 16; BC-KO DMSO:6; BC-KO MLN:8.

Article Snippet: Sheep polyclonal Alpha Tubulin antibody , Cytoskeleton , Cat#ATN02; RRID: AB_10708807.

Techniques: Imaging

Journal: iScience

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocyte meiotic spindle building

doi: 10.1016/j.isci.2024.110451

Figure Lengend Snippet: MTOC-AURKA partially rescued spindle phenotypes in ABC-KO oocytes (A) Schematic of the experimental design. ABC-KO oocytes microinjected with different AURKA fusions. Localization of AURKA is green. (B) Representative confocal images of Metaphase I ABC-KO oocytes expressing the indicated fusions. Non-injected ABC-KO oocytes were control. Oocytes were immunostained with TACC3 (magenta), tubulin (green), DAPI (blue). Localization of the targeted AURKA fusion is gray. Scale bar 10 μm. (C) Quantification of spindle and LISD parameters in (B). Gray dots: Non-injected oocytes; pink dots: WT-AURKA; blue dots: MTOC-AURKA + chromatin-AURKA. (D and E) Representative confocal images of Metaphase I ABC-KO oocytes expressing the indicated fusions immunostained with tubulin (green), DAPI (DNA) in (D) and PCNT (green), TACC3 (magenta), DAPI (DNA) in (E). Localization of the targeted AURKA fusion is gray. Scale bars: 5 μm. (F) Quantification of aMTOC parameters. (G) Quantification of spindle and LISD parameters. Gray dots: Non-injected oocytes; pink dots: WT-AURKA; green dots: MTOC-AURKA, and purple dots: chromatin-AURKA. (H) Live cell light-sheet imaging of WT and ABC-KO oocytes. ABC-KO oocytes express the indicated fusion, Sir-tubulin (green), DNA (magenta). (I) Time of MT nucleation and (J) time of spindle bipolarization (One-way ANOVA, ∗∗∗∗ p < 0.0001). Data are represented as mean ± SEM. (K) Spindle volume during meiotic maturation. Time stamp (h:min) is relative to GVBD. (L) Representative confocal image of ABC-KO oocytes from three mice at Metaphase I expressing the indicated Gfp fusions (gray). Oocytes were immunostained with tubulin (green) and DAPI (blue). Scale bar 10 μm. (M) Quantification of spindle volume in (L). (One-way ANOVA, ∗∗ p < 0.001; ∗∗∗ p < 0.001; ns = not significant). In brackets are the number of oocytes analyzed in at least 3 independent experiments. See also Figure S3–S6 .

Article Snippet: Sheep polyclonal Alpha Tubulin antibody , Cytoskeleton , Cat#ATN02; RRID: AB_10708807.

Techniques: Expressing, Injection, Control, Imaging

Journal: iScience

Article Title: Spatio-temporal requirements of Aurora kinase A in mouse oocyte meiotic spindle building

doi: 10.1016/j.isci.2024.110451

Figure Lengend Snippet:

Article Snippet: Sheep polyclonal Alpha Tubulin antibody , Cytoskeleton , Cat#ATN02; RRID: AB_10708807.

Techniques: Recombinant, Plasmid Preparation, Software