Journal: bioRxiv

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

doi: 10.1101/2024.04.01.587547

Figure Lengend Snippet: (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, immuno-stained 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: Primary antibodies and concentrations were used as follows: Pericentrin (Pcnt) (mouse, 1:100; BD Biosciences, #611814); TACC3 (Rabbit, 1:100; Novus Biologicals # NBP2-67671), Alpha Tubulin polyclonal (Sheep, 1:100; Cytoskeleton #ATN02); phosphorylated ABC (rabbit, 1:100; Cell Signaling Technology, #2914), phosphorylated CDC25B (1:100; Signalway Antibodies #11949); phosphorylated INCENP (Gift from Michael Lampson), Human anti-ACA (1:30, Antibodies Incorporated #15-234).

Techniques: Staining

Journal: bioRxiv

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

doi: 10.1101/2024.04.01.587547

Figure Lengend Snippet: (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, immuno-stained 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: Primary antibodies and concentrations were used as follows: Pericentrin (Pcnt) (mouse, 1:100; BD Biosciences, #611814); TACC3 (Rabbit, 1:100; Novus Biologicals # NBP2-67671), Alpha Tubulin polyclonal (Sheep, 1:100; Cytoskeleton #ATN02); phosphorylated ABC (rabbit, 1:100; Cell Signaling Technology, #2914), phosphorylated CDC25B (1:100; Signalway Antibodies #11949); phosphorylated INCENP (Gift from Michael Lampson), Human anti-ACA (1:30, Antibodies Incorporated #15-234).

Techniques: Staining

Journal: bioRxiv

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

doi: 10.1101/2024.04.01.587547

Figure Lengend Snippet: (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: Primary antibodies and concentrations were used as follows: Pericentrin (Pcnt) (mouse, 1:100; BD Biosciences, #611814); TACC3 (Rabbit, 1:100; Novus Biologicals # NBP2-67671), Alpha Tubulin polyclonal (Sheep, 1:100; Cytoskeleton #ATN02); phosphorylated ABC (rabbit, 1:100; Cell Signaling Technology, #2914), phosphorylated CDC25B (1:100; Signalway Antibodies #11949); phosphorylated INCENP (Gift from Michael Lampson), Human anti-ACA (1:30, Antibodies Incorporated #15-234).

Techniques: Imaging

Journal: bioRxiv

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

doi: 10.1101/2024.04.01.587547

Figure Lengend Snippet: (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 immuno-stained with TACC3 (magenta), tubulin (green), DAPI (blue). Localization of the targeted AURKA fusion is gray. (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, E) Representative confocal images of Metaphase I ABC-KO oocytes expressing the indicated fusions immuno-stained with tubulin (green), DAPI (DNA) in (D) and PCNT (green), TACC3 (magenta), DAPI (DNA) in (E). Localization of the targeted AURKA fusion is gray. Yellow arrow indicates MTOC-AURKA at kinetochores. Scale bar: 5µm. (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 expresses the indicated fusion, Sir-tubulin (green), DNA (magenta). (G) Time of MT nucleation and (H) 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. In brackets are the number of oocytes analyzed in at least 3 independent experiments. See also Figure S3-S6.

Article Snippet: Primary antibodies and concentrations were used as follows: Pericentrin (Pcnt) (mouse, 1:100; BD Biosciences, #611814); TACC3 (Rabbit, 1:100; Novus Biologicals # NBP2-67671), Alpha Tubulin polyclonal (Sheep, 1:100; Cytoskeleton #ATN02); phosphorylated ABC (rabbit, 1:100; Cell Signaling Technology, #2914), phosphorylated CDC25B (1:100; Signalway Antibodies #11949); phosphorylated INCENP (Gift from Michael Lampson), Human anti-ACA (1:30, Antibodies Incorporated #15-234).

Techniques: Expressing, Injection, Control, Staining, Imaging

Journal: bioRxiv

Article Title: Atlas of the Neuromuscular System in the Trachymedusa Aglantha digitale : Insights from the advanced hydrozoan

doi: 10.1101/772418

Figure Lengend Snippet: The structure of the umbrella body wall in Aglantha . α-Tubulin antibody (AB) is green, phalloidin is red, and nuclear DAPI staining is blue. ( a ) The outside layer of exumbrella is a layer of large epithelial cells, which borders are labeled by phalloidin. ( b ) The layer of circular striated muscles, which powerful contractions cause the propulsion movements of Aglantha . These muscles are parts of the myoepithelium and located close to the subumbrella surface. ( c ) The layer of striated muscles is brightly stained by phalloidin and can mask other structures. However, if striated muscles are cut and pulled aside, as indicated by arrows, additional details can be revealed. ( d ) Numerous individual smooth muscle fibers (arrows) in the body wall, which have the shape of a spindle. ( e ) At high magnification individual intracellular filaments, labeled by α-tubulin antibody, could be seen inside the cells (cell borders are indicated by arrows). ( f ) The velum at the bottom of the umbrella with a layer of circular muscles. Scale bars: a - 25 µm; b, d - 10 µm; c - 40 µm; e - 15 µm; f - 250 µm.

Article Snippet: Rat monoclonal anti-tyrosinated alpha-tubulin antibody is raised against yeast tubulin, clone YL1/2, isotype IgG2a (Serotec Cat # MCA77G; RRID: AB_325003).

Techniques: Staining, Labeling, Muscles

Journal: bioRxiv

Article Title: Atlas of the Neuromuscular System in the Trachymedusa Aglantha digitale : Insights from the advanced hydrozoan

doi: 10.1101/772418

Figure Lengend Snippet: Aglantha ’s manubrium attached to peduncle on one side, with mouth and four lips on the other side ( a , b ). In all images: α-tubulin antibody (AB) labeling is green; phalloidin - red, and DAPI - blue. ( c ) The inner surface of the lips is covered with nematocytes (cnidocytes), which are brightly labeled by α-tubulin AB (arrows). ( d ) Those nematocytes have the same structure as cnidocytes on the tentacles and have a single mechanosensory cilium (cnidocil) labeled by α-tubulin AB (arrows). The surrounding ring of microvilli is labeled by phalloidin (red). ( e ) The thin wall of a peduncle has fine muscle fibers labeled by phalloidin. ( f ) Closer to the umbrella top, the muscle fibers in the peduncle wall become shorter (arrows) and have a spindle-like shape similar to ( d , e ). Scale bars: a - 500 µm; b, e - 200 µm; c, f - 50 µm; d - 10 µm.

Article Snippet: Rat monoclonal anti-tyrosinated alpha-tubulin antibody is raised against yeast tubulin, clone YL1/2, isotype IgG2a (Serotec Cat # MCA77G; RRID: AB_325003).

Techniques: Labeling

Journal: bioRxiv

Article Title: Atlas of the Neuromuscular System in the Trachymedusa Aglantha digitale : Insights from the advanced hydrozoan

doi: 10.1101/772418

Figure Lengend Snippet: Neural plexus in the manubrium and gonads labeled by α-tubulin AB (green). Phalloidin is red, while nuclear DAPI is blue ( a , b ). There is a dense neural plexus in the ectodermal layer of the manubrium in Aglantha . ( c ) Higher resolution image shows individual neural cell bodies (arrows) and multiple processes connecting and forming a network-like mesh. ( d ) The nerve processes are next to the nematocytes on the inner surface of the lips (arrows). ( e , f ) Neural-like cells labeled by α-tubulin AB form a network on the surface of the gonads (arrows). Scale bars: a, e - 100 µm; b - 40 µm; c - 10 µm; d - 20 µm; f - 50 µm.

Article Snippet: Rat monoclonal anti-tyrosinated alpha-tubulin antibody is raised against yeast tubulin, clone YL1/2, isotype IgG2a (Serotec Cat # MCA77G; RRID: AB_325003).

Techniques: Labeling

Journal: bioRxiv

Article Title: Atlas of the Neuromuscular System in the Trachymedusa Aglantha digitale : Insights from the advanced hydrozoan

doi: 10.1101/772418

Figure Lengend Snippet: Motor giant axons in Aglantha labeled by α-tubulin AB (green). Phalloidin labels striated muscles in the umbrella wall (red), while nuclear DAPI is blue. ( a ) Each giant motor axon runs across the entire umbrella from the margin to the apex. In the margin, it divides into a few short branches (arrows) upon entering the nerve ring that circumvents the velum. ( b ) Higher magnification shows that α-tubulin AB label numerous thin filaments inside a giant axon, which represent long α-tubulin threads inside the giant syncytium. ( c ) Along the entire length of motor giants, there are more or less evenly spread lateral neural branches (arrows) running perpendicular to the giants and in parallel to the striated muscle fibers. ( d ) These lateral projections look like individual branches of the motor giants even at high magnification (arrows). However, the work by identified them as separate lateral neurons. ( e ) Lateral neurons branch extensively in the striated muscle layer. ( f ) Lateral neurons from neighboring motor giants (1 and 2) have overlapping innervation fields and even connect to each other. Abbreviations: MGA - motor giant axon; INR - inner nerve ring; ONR - outer nerve ring. Scale bars: a - 50 µm; b, d - 25 µm; c, e, f - 100 µm.

Article Snippet: Rat monoclonal anti-tyrosinated alpha-tubulin antibody is raised against yeast tubulin, clone YL1/2, isotype IgG2a (Serotec Cat # MCA77G; RRID: AB_325003).

Techniques: Labeling, Muscles

Journal: bioRxiv

Article Title: Atlas of the Neuromuscular System in the Trachymedusa Aglantha digitale : Insights from the advanced hydrozoan

doi: 10.1101/772418

Figure Lengend Snippet: Large axon running along the radial digestive canal. α-Tubulin AB labeling is green, while nuclear DAPI is blue. ( a ) The radial digestive canal is outlined by black arrows in this image obtained with a transmitted light channel (TD). The axon (white arrows) in the middle of the canal is labeled by α-tubulin AB. ( b ) The long axon is produced by a single neuron (arrow). ( c ) This neuron (arrow) is tripolar with two larger branches running in opposite directions. Scale bars: a, b - 100 µm; c - 40 µm.

Article Snippet: Rat monoclonal anti-tyrosinated alpha-tubulin antibody is raised against yeast tubulin, clone YL1/2, isotype IgG2a (Serotec Cat # MCA77G; RRID: AB_325003).

Techniques: Labeling, Produced

Journal: bioRxiv

Article Title: Atlas of the Neuromuscular System in the Trachymedusa Aglantha digitale : Insights from the advanced hydrozoan

doi: 10.1101/772418

Figure Lengend Snippet: The nerve ring labeled by α-tubulin AB (green). Phalloidin is red; nuclear DAPI is blue. ( a ) The nerve ring circumvents the velum at the margin of the umbrella (arrows). ( b ) Crossing the nerve ring ( NR ) throughout its entire length, numerous short ‘cords’ are brightly labeled by α-tubulin AB - two on both sides of each tentacle base. Those cords are composed of sensory cells that form the comb pads on their wide side (arrows). ( c ) The nerve ring consists of two rings - outer nerve ring ( ONR ) and the inner nerve ring ( INR ), which can be seen at higher magnification. The outer nerve ring ( ONR ) contains a giant axon. ( d ) There are many thin processes originating from the ring that innervate the neighboring area of the body wall and form a basal plexus (arrows). ( e ) We have identified eight evenly spread bush-like structures attached to the nerve ring that consisted of multiple branches labeled by α-tubulin AB - also indicated as bls in ( b ). These structures were also characterized by several rows of tightly packed nuclei at different levels (arrows). ( f ) The addition of a transmitted light channel (TD) showed that at the base of each “bush” there was a statocyst. Scale bars: a - 500 µm; b, d - 100 µm; c, e, f - 25 µm.

Article Snippet: Rat monoclonal anti-tyrosinated alpha-tubulin antibody is raised against yeast tubulin, clone YL1/2, isotype IgG2a (Serotec Cat # MCA77G; RRID: AB_325003).

Techniques: Labeling

Journal: bioRxiv

Article Title: Atlas of the Neuromuscular System in the Trachymedusa Aglantha digitale : Insights from the advanced hydrozoan

doi: 10.1101/772418

Figure Lengend Snippet: Sensory comb pads labeled by α-tubulin AB (green) and phalloidin (red), while nuclear DAPI is blue. ( a ) Comb pads are located on the wider end of the short and thick cords brightly labeled by α-tubulin AB. Those cords extend from the tentacle base, cross the nerve ring ( NR ) and widen into the actual comb pad at their end (arrows). ( b ) The microvilli surrounding the base of long comb pads cilia are brightly labeled by phalloidin. Each ring of microvilli looks like a short spike or thorn (arrows). ( c ) The long cilia themselves are not labeled by α-tubulin AB or phalloidin and could be seen only with a transmitted light channel (arrows). ( d ) Comb pads vary in size with smaller pads having only 15-20 sensory cilia with their microvilli labeled by phalloidin (arrows). ( e ) Comb pad cords (arrows) abruptly end on both sides of a muscle bundle connecting each tentacle base to the umbrella. ( f ) While crossing the nerve ring, comb pad cords run very close to the ring giant axon ( RGA ) almost making contact. The entire thickness of this optical section is only 5 µm. Note also that tentacle base giant axons ( TBGA ) contact the ring giant axon ( RGA ). (g) Short, thin branches (arrows) are connecting the tentacle base giant axons ( TBGA ) with the comb pad cords. Scale bars: a - 25 µm; b, c - 15 µm; d - 10 µm; e, f, g - 20 µm.

Article Snippet: Rat monoclonal anti-tyrosinated alpha-tubulin antibody is raised against yeast tubulin, clone YL1/2, isotype IgG2a (Serotec Cat # MCA77G; RRID: AB_325003).

Techniques: Labeling

Journal: bioRxiv

Article Title: Atlas of the Neuromuscular System in the Trachymedusa Aglantha digitale : Insights from the advanced hydrozoan

doi: 10.1101/772418

Figure Lengend Snippet: Innervation of the tentacles. α-Tubulin IR is green, phalloidin is red, while nuclear DAPI is blue. ( a ) Numerous thin and long tentacles (arrows) are attached to the margin of the umbrella in Aglantha . ( b ) Each tentacle has a distinct short tentacle base ( TB ). Two short cords labeled by α-tubulin AB, which form the sensory comb pads on one side, approach the base of each tentacle on the other side (arrows). ( c ) α-Tubulin AB also label two giant axons (arrows) that cross the base of each tentacle. ( d ) Two tentacle base giant axons (arrows) connect to the nerve ring ( NR ), making a visible contact with the ring giant axon ( RGA ). ( e ) The tentacle base giant axons (arrows) run across the base of the tentacle and form a ring-like connection to each other (arrowhead). ( f ) The ring, formed by tentacle base giant axons, gives rise to three axons (arrows) that run through the entire length of each tentacle. One axon (in the middle) is thicker than the other two and might correspond to the tentacle giant axon. ( g ) Close to their base, three axons produce a grid-like structure of connecting neural branches (arrows). ( h ) Further down, three axons (arrows) are observed crossing the entire length of the tentacles. ( i ) Fewer connecting branches (arrows) in the middle section of a tentacle. Scale bars: a - 200 µm; b, c, h - 100 µm; d - 30 µm; e - 40 µm; f - 20 µm; g - 50 µm; i - 25 µm.

Article Snippet: Rat monoclonal anti-tyrosinated alpha-tubulin antibody is raised against yeast tubulin, clone YL1/2, isotype IgG2a (Serotec Cat # MCA77G; RRID: AB_325003).

Techniques: Labeling

Journal: bioRxiv

Article Title: Atlas of the Neuromuscular System in the Trachymedusa Aglantha digitale : Insights from the advanced hydrozoan

doi: 10.1101/772418

Figure Lengend Snippet: Muscles and ‘sensory’ cilia in the tentacles, labeled by phalloidin (red), α-tubulin AB (green) and nuclear DAPI (blue). ( a ) Tentacles are very muscular and intensively stained by phalloidin (red). There are two main groups of muscle fibers in each tentacle: circular muscle fibers (arrows) and long longitudinal muscle fibers. ( b , c ) Phalloidin also labels groups of muscle fibers at the very edge of the umbrella between the tentacles (arrows). ( d , e ) In addition, phalloidin brightly stained several rows of very short but pronounced ‘thorns’ or ‘spikes’ at the base of each tentacle (arrows). Each spike represented a compact group of short microvilli, which could be seen more clearly at SEM images . ( f ) The ring of these microvilli surrounded the long sensory cilia (arrows), which could be seen only with a transmitted light channel (TD) since they were not labeled by α-tubulin AB or phalloidin. ( g ) Neural connections to the sensory cilia at the tentacle base could be mediated by neurites (arrows) running along the cilia row and labeled by α-tubulin AB (green). ( h ) A powerful bundle of muscles (arrowhead) in the umbrella margin connects the base of each tentacle to deeper layers up to the ring nerve. Note the distant ends (arrows) of the sensory comb pads cords brightly labeled by α-tubulin AB. ( i ) A row of cell-like structures (arrows) is always labeled by α-tubulin AB at the tentacle base, where it connects to the umbrella margin. Scale bars: a, c, d, h - 40 µm; b - 100 µm; e - 15 µm; f - 10 µm; g, i - 25 µm.

Article Snippet: Rat monoclonal anti-tyrosinated alpha-tubulin antibody is raised against yeast tubulin, clone YL1/2, isotype IgG2a (Serotec Cat # MCA77G; RRID: AB_325003).

Techniques: Muscles, Labeling, Staining

Journal: bioRxiv

Article Title: Atlas of the Neuromuscular System in the Trachymedusa Aglantha digitale : Insights from the advanced hydrozoan

doi: 10.1101/772418

Figure Lengend Snippet: Nematocytes (cnidocytes) on the tentacles of Aglantha , labeled with α-tubulin AB (green), phalloidin (red) and nuclear DAPI (blue). ( a ) Nematocytes are labeled by α-tubulin AB (green). They are grouped in clusters (arrows) along the tentacles, which look like evenly spread spherical balls and remind of a strand of beads. ( b , c ) There are significantly more nematocytes at the tentacle end, and fewer closer to the tentacle base. Note the muscle bundles in the tentacles labeled by phalloidin (red). ( d ) Each intact cnidocyte is a cell with a single nucleus that has a mechanosensory single cilium (cnidocil) labeled by α-tubulin AB (arrows) on the top. ( e , f ) Phalloidin also labeled a ring of microvilli (arrows) at the base of the cnidocil cilium. In some preparations, we could count seven microvilli grouped around α-tubulin AB labeled cilium in the center ( f ). Scale bars: a, b - 50 µm; c - 25 µm; d, f - 10 µm; e - 20 µm.

Article Snippet: Rat monoclonal anti-tyrosinated alpha-tubulin antibody is raised against yeast tubulin, clone YL1/2, isotype IgG2a (Serotec Cat # MCA77G; RRID: AB_325003).

Techniques: Labeling

Journal: bioRxiv

Article Title: Atlas of the Neuromuscular System in the Trachymedusa Aglantha digitale : Insights from the advanced hydrozoan

doi: 10.1101/772418

Figure Lengend Snippet: RFamide immunoreactivity (RFa-IR) in the umbrella margin and tentacles (labeled in red; while α-tubulin AB immunostaining is green). ( a ) Overview of immunoreactivity in the umbrella margin, from the nerve ring ( NR ) to the tentacle bases ( TB ). Arrows indicate some of the identified RFa-IR neurons. Note the tracts of RFamide-IR fibers running from the nerve ring to the tentacles. ( b ) Each tentacle base contains a plexus of RFa-immunoreactive processes and two larger neurons (arrows), which were identified as star cells by . (b) RFa immunoreactive cells in the tentacles (arrows). ( d ) Many round-shaped cells (arrows) are located along the outer nerve ring ( ONR ) through its entire length, along with numerous RFa immunoreactive processes inside the nerve ring. ( e , f ) A pair of large RFa immunoreactive neurons (arrows) has been identified next to each tentacle base giant axon (arrowheads) not far from where they enter the outer nerve ring ( ONR ). The thickest branches of these neurons (red) follow the tentacle base giant axons (green), while several smaller branches run in parallel contributing to the tract of IR fibers to the tentacles. Neurons in each pair are connected with a short connective. RFa immunoreactive neural processes (red) run along the thick comb pad cords (green). Note that RFa IR does not co-localize with α-tubulin IR, suggesting that α-tubulin AB do not label all neural elements in Aglantha . Scale bars: a - 100 µm; b, c - 40 µm; d, e - 30 µm; f - 10 µm.

Article Snippet: Rat monoclonal anti-tyrosinated alpha-tubulin antibody is raised against yeast tubulin, clone YL1/2, isotype IgG2a (Serotec Cat # MCA77G; RRID: AB_325003).

Techniques: Labeling, Immunostaining

Journal: bioRxiv

Article Title: Atlas of the Neuromuscular System in the Trachymedusa Aglantha digitale : Insights from the advanced hydrozoan

doi: 10.1101/772418

Figure Lengend Snippet: RFamide immunoreactivity in the manubrium and mouth (labeled in red; while α-tubulin AB immunostaining is green). ( a ) Manubrium and mouth with four lips. Arrowheads point to the RFa immunoreactive tracts, which run across the entire manubrium from peduncle to the tip of a lip. ( b ) Higher magnification of a mouth end of the manubrium. Arrows indicate some of the RFa immunoreactive neurons. ( c ) RFa immunoreactive plexus (red) at the tip of a lip. ( d ) The central part of the manubrium, where the narrow tracts expand into much wider stretches of RFa immunoreactive fibers (arrowheads). ( e , f ) RFa immunoreactive neurons (arrows) with their processes. Note that those neurons and their processes (red) do not overlap with α-tubulin immunoreactive plexus (green). Scale bars: a - 200 µm; b, d - 100 µm; c - 30 µm; e, f - 40 µm.

Article Snippet: Rat monoclonal anti-tyrosinated alpha-tubulin antibody is raised against yeast tubulin, clone YL1/2, isotype IgG2a (Serotec Cat # MCA77G; RRID: AB_325003).

Techniques: Labeling, Immunostaining

Journal: PLoS Genetics

Article Title: Topoisomerase II Is Required for the Proper Separation of Heterochromatic Regions during Drosophila melanogaster Female Meiosis

doi: 10.1371/journal.pgen.1004650

Figure Lengend Snippet: DNA is labeled with DAPI (blue), chromatin is labeled with an antibody recognizing H3S10p (green), and the spindle is labeled with an antibody recognizing α-tubulin (red). (A) Top2 RNAi /+ control oocyte with achiasmate 4 th chromosomes that have moved towards the spindle poles. Arrowhead points to the H3S10p-labeled heterochromatic DNA thread emanating from the achiasmate 4 th chromosome. (B–D) Shown are examples of Top2 RNAi /matαGAL oocytes containing abnormal DNA projections that do not appear to connect chromosomes (arrowheads). Images are projections of partial Z-stacks. Scale bars are 5 microns.

Article Snippet: The primary antibody used for Western blot was rabbit anti-Top2 at a dilution of 1∶5000 and α-tubulin (Serotec) at a dilution of 1∶5000.

Techniques: Labeling, Control

Journal: PLoS Genetics

Article Title: Topoisomerase II Is Required for the Proper Separation of Heterochromatic Regions during Drosophila melanogaster Female Meiosis

doi: 10.1371/journal.pgen.1004650

Figure Lengend Snippet: DNA is labeled with DAPI (blue), heterochromatin is labeled with an antibody recognizing histone 3 trimethylated on lysine 9 (H3K9me3) (green), and the spindle is labeled with an antibody recognizing α-tubulin (red). (A) Top2 RNAi /+ control oocyte with achiasmate 4 th chromosomes that have moved towards the spindle poles. The 4 th chromosomes and the centromeric regions of the chromosomes are labeled with H3K9me3 and are oriented towards opposite spindle poles. (B–C) Top2 RNAi /matαGAL oocytes containing abnormal DNA projections that are labeled with the H3K9me3 antibody. Additionally, the H3K9me3 antibody localization in the chromosome mass of both oocytes is not oriented towards opposite spindle poles. Images are projections of partial Z-stacks. Scale bars are 5 microns.

Article Snippet: The primary antibody used for Western blot was rabbit anti-Top2 at a dilution of 1∶5000 and α-tubulin (Serotec) at a dilution of 1∶5000.

Techniques: Labeling, Control

Journal: PLoS Genetics

Article Title: Topoisomerase II Is Required for the Proper Separation of Heterochromatic Regions during Drosophila melanogaster Female Meiosis

doi: 10.1371/journal.pgen.1004650

Figure Lengend Snippet: DNA is labeled with DAPI (blue) and spindles are labeled with an antibody recognizing α-tubulin (yellow). (A and B) Top2 RNAi /+ embryos displaying normal development. Boxed regions are shown magnified below each image. (C and D) Embryos from Top2 RNAi/ matαGAL mothers with two nuclei indicating proper fertilization but an arrest in meiosis I. 17/20 embryos from Top2 RNAi /matαGAL mothers contained only 2 nuclei. Of the remaining 3 embryos, one contained only a single nucleus and 2 appeared to undergo an aberrant division. Arrowheads indicate regions magnified below both images. Images are projections of partial Z-stacks. Scale bars are 20 microns.

Article Snippet: The primary antibody used for Western blot was rabbit anti-Top2 at a dilution of 1∶5000 and α-tubulin (Serotec) at a dilution of 1∶5000.

Techniques: Labeling

Journal: Osteoarthritis and cartilage

Article Title: SOX9 protein is stabilized by TGF-β and regulates PAPSS2 mRNA expression in chondrocytes

doi: 10.1016/j.joca.2016.10.007

Figure Lengend Snippet: ATDC5 cells were transfected with Sox9 siRNA, Smad2/3 siRNA, or control non-specific (NS) siRNA. Sox9 mRNA expression was significantly reduced in cells expressing Sox9 siRNA (A). Acan mRNA was significantly reduced in the presence of Sox9 siRNA (B). Cells containing NS siRNA or Sox9 siRNA were treated with TGF-β1 and expression of Papss2 mRNA was measured by qPCR (C). In A–C * = p < 0.05 REST, n = 5. Western blots showed Smad2/3 protein levels were reduced in the presence of Smad2/3 siRNA, α-Tubulin was used as a loading control (n = 6) (D). Acan mRNA was significantly down-regulated in the Smad2/3 siRNA-transfected cells compared to cells containing NS siRNA (REST, * = p < 0.05, n = 2) (E). Cells containing NS or Smad2/3 siRNA siRNA were treated with TGF-β1 and Papss2 mRNA was, measured by qPCR (REST, * = p <0.05, n.s = not-significant n = 4) (F).

Article Snippet: To assess whether equivalent amounts of protein were loaded in all wells, either an anti-GAPDH primary antibody (1:1000, Santa Cruz Biotechnology sc-25778) or anti-α-Tubulin primary antibody (1:2500, Rockland Immunochemicals #200-301-880) were used.

Techniques: Transfection, Expressing, Western Blot

Journal: Osteoarthritis and cartilage

Article Title: SOX9 protein is stabilized by TGF-β and regulates PAPSS2 mRNA expression in chondrocytes

doi: 10.1016/j.joca.2016.10.007

Figure Lengend Snippet: In A, mRNA was collected from bovine chondrocytes treated with vehicle or TGF-β1 for the indicated amounts of time. SOX9 mRNA levels were determined by qPCR (REST, no statistically significant differences, n = 5). In B, protein was collected from bovine chondrocytes that were treated with either vehicle or TGF-β1 for 4 or 6 hours. SOX9, pSMAD3, and SMAD2/3 protein levels were determined by Western blot (n = 3). α-Tubulin was used as a loading control. In C, new protein synthesis was inhibited with cycloheximide. Cells were subsequently treated with either vehicle or TGF-β1 for up to 8 hours. Protein was isolated at specified time points, and the levels of SOX9 protein were determined by Western blot (n = 3). pSMAD3 = phosphorylated SMAD3, CHX = cycloheximide.

Article Snippet: To assess whether equivalent amounts of protein were loaded in all wells, either an anti-GAPDH primary antibody (1:1000, Santa Cruz Biotechnology sc-25778) or anti-α-Tubulin primary antibody (1:2500, Rockland Immunochemicals #200-301-880) were used.

Techniques: Western Blot, Isolation

Journal: bioRxiv

Article Title: Precise control of microtubule disassembly in living cells

doi: 10.1101/2021.10.08.463668

Figure Lengend Snippet: a . COS7 cells co-transfected with EMTB-YFP-CIBN and mCh-Cry2 (red) were incubated with SPY650-tubulin (SPY650-tub) to visualize MTs. The cells were illuminated by blue light within a specified region (indicated by the dotted circle) for the indicated time period. Scale bar, 10 μm. b . The normalized intensity of mCh-Cry2 at MTs in illuminated regions (red) and non-illuminated regions (green). n = 6 cells from three independent experiments. c . COS7 cells co-transfected with EMTB-YFP-CIBN and dNSpastinXXX-mCh-Cry2 were incubated with SPY650-tubulin to visualize MTs. The cells were illuminated by blue light as in a . Scale bar, 10 μm. d . The normalized intensity of dNSpastinXXX-mCh-Cry2 and mCh-Cry2 at MTs and the normalized area of SPY650-tubulin in illuminated regions and non-illuminated regions. n = 6 and 6 cells in dNSpastinXXX-mCh-Cry2 and mCh-Cry2 group, respectively, from three independent experiments. Data are shown as the mean ± S.E.M.

Article Snippet: Before imaging, cells were incubated with SPY650-tubulin (1000-fold dilution; Spirochrome) at 37°C for 1 hr.

Techniques: Transfection, Incubation

Journal: bioRxiv

Article Title: Precise control of microtubule disassembly in living cells

doi: 10.1101/2021.10.08.463668

Figure Lengend Snippet: COS7 cells were co-transfected with EMTB-YFP-CIBN and mCh-Cry2 (red), and MTs were labeled with SPY650-tubulin (SPY650-tub; black). Illumination with blue light for the indicated period of time triggered rapid recruitment of mCh-Cry2 onto MTs in the light-illuminated region (dotted circle). The MTs, however, remained intact after recruitment of mCh-Cry2. Scale bar, 10 μm.

Article Snippet: Before imaging, cells were incubated with SPY650-tubulin (1000-fold dilution; Spirochrome) at 37°C for 1 hr.

Techniques: Transfection, Labeling