Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) Left, the crystal structure of the Aurora A kinase domain in an active conformation, shown in grey ribbons representation, adopts the canonical kinase domain fold with N-terminal and C-terminal lobes. The ATP binding pocket containing bound AMP-PNP, shown in stick representation (with carbons colored dark grey, oxygens red, and phosphorus orange) is located within the active site cleft in between the two lobes. Side chains of Cys247, Cys290, and phosphorylated Thr288 are also shown in stick representation. Right, the crystal structure of the Aurora A kinase domain obtained with cacodylate buffer, shown in blue ribbons representation, depicting covalent modification of Cys247 and Cys290 (shown in stick representation with cyan carbons, overlayed with transparent space filled rendering) and a large displacement of the activation segment (shown in orange). TPX2 residues 7–20 are shown in yellow. (B) The crystal structure of the activation segment-swapped dimer of the cacodylate-modified Aurora kinase domain. Monomers are colored blue and purple and shown as ribbons representations, with a cartoon indicating the relative orientation of each monomer in the upper right of the structure. The activation segment is orange. Symmetry-related kinase domain monomers within the crystal structure can be seen in a dimeric arrangement with their activation segments exchanged between the monomers at the dimer interface. The monomers are oriented with their N-terminal lobes pointing in near-orthogonal directions, as indicated by the dotted axis lines in the cartoon representation.

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Binding Assay, Modification, Activation Assay

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) Superposition of the active sites in the cacodylate-modified Aurora A kinase domain (blue and cyan) with the corresponding unmodified structure (grey and dark grey) shows spatial overlap between Phe275 in the active DFG-in conformation (dark grey, in stick representation) and the dimethyl arsenic adduct of the cacodylate-modified Cys247 (cyan and purple in stick representation, overlayed with transparent space filled rendering). AMP-PNP in the active structure (stick representation with dark grey carbons) spatially overlaps with Phe275 in the inactive cacodylate-modified DFG-out structure (cyan), and the side chain of Glu181 is also displaced in the cacodylate-modified structure (cyan) relative to the unmodified structure (dark grey). (B) The displacement of the Glu181 side chain expands the ATP-binding pocket, allowing an oxidized DTT molecule to occupy this space in the cacodylate-modified Aurora A structure. Superposition of the previous structure of the Aurora A kinase domain in complex with a 5-aminopyrimidinyl quinazoline inhibitor (beige and yellow; PDB code 2C6E) shows how this expanded ATP-binding pocket accommodates existing Aurora A-selective inhibitors. (C) Schematic of the experiment in which an Aurora A kinase domain construct containing a single cysteine (Cys247) was used for a mass spectrometry-based high throughput tethering screen of 880 disulfide-containing compounds (listed in data file S1) to identify additional covalent modifiers of Cys247. Each compound was incubated separately with Aurora A kinase domain, to allow thiol-disulfide exchange with the Cys247 side chain thiol in the presence of ß-mercaptoethanol, a non-specific disulfide reducing agent. Stable covalent labeling of Cys247 under these mildly reducing conditions requires additional stabilizing contacts between the particular compound and residues in Aurora A in close proximity to Cys247. These stable disulfide adducts were then detected by an increased total mass of the protein using mass spectrometry (data file S1).

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Modification, Binding Assay, Construct, Mass Spectrometry, High Throughput Screening Assay, Incubation, Labeling

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A and B) Structures of the Aurora A kinase domain modified with compounds 7–80 (A) and 8–34 (B) at Cys290 show an activation segment-swapped dimer, in which the monomers are oriented with their N-terminal lobes pointing in the same direction, as indicated by the dotted axis lines in the cartoon representation in the center bottom. Monomers are colored blue and purple and shown as ribbons representations. The activation segment is orange. This dimer configuration is distinct from the near orthogonal arrangement seen in the cacodylate-modified structure (Fig. 1B). (C) Superposition of the active site regions of the 7–80 modified Aurora A structure with the Akt kinase domain (dark green and chartreuse) in complex with AMP-PNP and a GSK3ß substrate peptide (beige and yellow) from PDB code 1O6L. The active site of one monomer of the 7–80 modified Aurora A is shown in blue and cyan, with the Thr288/Cys290 activation loop from the other monomer shown in purple and magenta. Sulfur atoms of Cys290 are colored bright green. (D and E) Structure of the 7–80-modified structure, superimposed and contrasted with each of the monomers from a structure of a fully reduced and unphosphorylated Aurora A kinase domain in a similar dimer configuration (PDB code 4C3P). Large differences in the positioning of the phopsho acceptor residue (Thr288) and of the catalytic base (Asp256) in the 4C3P structure (emphasized with black outlines) can be seen between the 7–80-modified and the unmodified structures.

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Modification, Activation Assay

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) The structure shows an activation segment-swapped dimer with the disulfide bond formed between Cys290 in each monomer (with sulfur atoms colored green) at the center of the dimer interface. Molecules are shown in cartoon representation with the monomers colored blue and magenta, and with a cartoon representation shown above the structure indicating the relative orientations of the monomers. (B) The cacodylate-modified Aurora A kinase domain dimer in an identical orientation as in (A), shows a similar overall dimer configuration but with conformational differences in the activation segments [green in (B) vs orange in (A)]. (C) Monomers of Aurora A from (A) and (B) are shown superimposed. The DFG-in active conformation of a monomer of the Aurora A kinase domain disulfide homodimer (Phe shown in stick representation, colored cyan) contrasts with the inactive DFG-out conformation of the cacodylate-modified kinase domain (colored yellow), and also with the inactive DFG-out conformation of the CoAlated disulfide-linked dimer structure (Fig. 6E).

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Activation Assay, Modification

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) Sequence alignment of human Aurora A (residues 246–293) with X. laevis Aurora A (residues 253–300). The highlighted amino acid residues denote residues mutated in the constructs used in the assays described in the remainder of this figure (B to D). (B to D) Xenopus egg extracts were used for xAurora A activation assays to examine the requirement for the activation loop cysteine (Cys297) of xAurora A in autophosphorylation. Endogenous xAurora A was depleted from Xenopus egg extracts using an immobilized xAurora A-binding fragment of xCEP192. Wild-type and mutant xAurora A constructs were then added to this depleted extract and assayed for activation by addition of either sperm nuclei as a source of centrosomes (B) or by addition of an antibody to xAurora A (C). In (D), xAurora A activation was also assayed using undepleted Xenopus egg extract and selective activation of exogenous wild-type and mutant FLAG-tagged xAurora A constructs using an antibody to FLAG. To the right of each experimental schematic, total and autophosphorylated (pThr295) Aurora A was detected by Western blotting. Blots in (B and D) are representative of 5 independent experiments, and the blots in (C) are representative of 3 independent experiments.

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Sequencing, Construct, Activation Assay, Binding Assay, Mutagenesis, Western Blot

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) Experimental schematic of the creation of stable HeLa cell lines by incorporating a doxycycline-inducible shRNA against endogenous Aurora A and transfected with or without shRNA-resistant, FLAG-tagged, wild-type and mutant Aurora A constructs driven by a native Aurora A promoter fragment. Following induction of shAurora A, the cells were nocodazole arrested. (B) Western blotting to assess Aurora A autophosphorylation at pThr288 in the nocodazole-arrested lysates described in (A). Total Aurora A and β-tubulin were blotted for reference. Blots are representative of 2 independent experiments.

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: shRNA, Transfection, Mutagenesis, Construct, Western Blot

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) Western blotting for Aurora A autophosphorylation at pThr288 to assess xAurora A activation in Xenopus egg extracts supplemented with demembranated sperm nuclei and exposed to DTT (or buffer, control) for the indicated times. Blots are representative of 4 independent experiments. (B) Schematic for Aurora A kinase domain constructs CoAlated on Cys290 and crystallized in the presence of AMP-PNP. (C) Structure of a wild type Aurora A kinase domain construct CoAlated on Cys290 shows an activation segment-swapped dimer. Monomers are colored blue and purple and shown as ribbons representations, with a cartoon shown above the structure. The activation segments (orange and red) are swapped between the monomers. The CoA adduct of one monomer is bound in the ATP-binding of pocket of the opposing monomer. No electron density was observed for the TPX2 fragment fused to the N-terminus of the Aurora A kinase domain construct used to determine this structure. (D) Structure of a single-cysteine human Aurora A kinase domain construct CoAlated on Cys290 and crystallized in complex with AMP-PNP shows an activation segment-swapped dimer with monomers oriented with their N-terminal lobes pointing in near orthogonal directions (dotted axis lines in the cartoon representation shown in the upper right of the panel), in contrast to the monomer orientation shown in panel C. Monomers are colored blue and purple and shown as ribbons representations. The activation segment is orange. (E) The CoAlated Aurora A kinase domain dimer from (D), with the activation segments colored orange and the DFG phenylalanine in cyan, is shown superimposed on the cacodylate-modified dimer, colored yellow/gold with activation segments colored green. Both structures show a catalytically inactive DFG-out activation segment conformation. (F) A 2mFo-DFc map contoured at 0.5σ shows electron density consistent with a subpopulation of the Aurora A kinase domain molecules in the crystal containing a Cys290-Cys290 symmetric disulfide.

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Western Blot, Activation Assay, Construct, Binding Assay, Modification

Journal: Science signaling

Article Title: Redox priming promotes Aurora A activation during mitosis

doi: 10.1126/scisignal.abb6707

Figure Lengend Snippet: (A) Catalytically active Aurora A kinase domain constructs treated with the disulfide-promoting Ellman’s reagent and incubated with ATP show robust Thr288 autophosphorylation as assayed by Western blotting. Phosphorylation of Thr288 is detected in the bands corresponding to dimers for both the wild type and the C247V + C319V mutant construct. Inclusion of DTT in the kinase assay abrogates Thr288 phosphorylation. Blots are representative of 2 independent experiments. (B) Upper panel, purification scheme for disulfide-linked heterodimers containing an MBP-tagged kinase dead and untagged catalytically active Aurora A kinase domain. Lower panel, following incubation with ATP, the samples were analyzed by SDFS-PAGE under non-reducing or reducing conditions, and probed for autophosphorylation by Western blotting. Blots are representative of 8 independent experiments. (C) Proposed model of redox and dimerization-dependent activation of Aurora A. We posit that increased protein cysteine oxidation during mitosis results in increased levels of disulfide modifications of proteins, such as CoAlation of Aurora A. Clustering of Aurora A molecules upon centrosomal recruitment promotes dimerization and thiol-disulfide exchange between kinase domains to form a disulfide homodimer that facilitates autophosphorylation. Resolution of the disulfide homodimer releases activated (pThr288) Aurora A monomers.

Article Snippet: Total xAurora A protein and pThr 295 were detected by Western blotting with a rabbit polyclonal xAurora A antibody and a rabbit monoclonal phospho-Aurora A [human pT288 / Xenopus pT295) antibody (D13A11, Cell Signaling Technology), respectively], and IRDye fluorescent secondary antibodies (LI-COR Biosciences).

Techniques: Construct, Incubation, Western Blot, Mutagenesis, Kinase Assay, Purification, Activation Assay

Journal: The Journal of Biological Chemistry

Article Title: LAT1 supports mitotic progression through Golgi unlinking in an amino acid transport activity-independent manner

doi: 10.1016/j.jbc.2024.107761

Figure Lengend Snippet: LAT1 promotes Golgi unlinking along with Aurora A recruitment to the centrosomes . A , HeLa S3 cells were fixed with formaldehyde and stained for LAT1 ( green ) and TGN46 ( red ) or calnexin ( red ). Representative images are shown. Scale bar, 10 μm. B–E , HeLa S3 cells were transfected with control siRNA (siControl) or LAT1-targeting siRNAs (siLAT1#1 and #2). At 19 h after siRNA transfection, the cells were treated with 4 mM thymidine for 19 h and washed with PBS(−). B and C , the cells were cultured for a further 9 h or 10 h in siControl or siLAT1 cells, respectively, to analyze the Golgi structure in prophase. The cells were fixed with MeOH and stained for TGN46 ( gray or green ) and DNA ( red ). B , representative z-stack images are shown, and Golgi objects based on TGN46 staining are numbered (see “ ”). Scale bar, 10 μm. C , the number of Golgi object within a cell was measured and plotted as the mean ± SD from a representative experiment of two independent experiments (n = 40). Statistical analysis was performed using Welch’s ANOVA ( F = 319, p = 0.000), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). D and E , The cells were cultured for a further 9.5 h or 10.5 h in siControl or siLAT1 cells, respectively, to analyze Aurora A recruitment in prometaphase cells. Then, the cells were fixed with MeOH and stained for Aurora A ( green ), phospho-Aurora A (pT288) ( red ), and DNA ( cyan ). D , representative z-stack images are shown. Scale bar, 10 μm. E , the fluorescence intensity of Aurora A ( left ) or phospho-Aurora A (pT288) ( right ) at centrosomes in prometaphase per cell was measured (see “ ”), and the average between the two centrosomes was plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). Statistical analysis was performed using Welch’s ANOVA ( F = 99.8, p = 0.000 in the left panel ; F = 88.6, p = 0.000 in the right panel ), and asterisks indicate significant differences (Games–Howell test, ∗∗∗ p < 0.001). F and G , HeLa S3 cells were treated with DMSO or 30 μM SP600125 for 2 h. F , the cells were fixed with MeOH and stained for TGN46 and DNA. The number of Golgi objects within a cell was measured and plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). G , the cells were fixed with MeOH and stained for Aurora A, phospho-Aurora A (pT288), and DNA. The fluorescence intensity of Aurora A ( left ) or phospho-Aurora A (pT288) ( right ) at centrosomes in prometaphase per cell was measured, and the average between the two centrosomes was plotted as the mean ± SD from a representative experiment of two independent experiments (n = 20). Asterisks indicate significant differences [Student’s t test in panel ( F and G ), ∗∗ p < 0.01; ∗∗∗ p < 0.001].

Article Snippet: The following primary antibodies were used for immunofluorescence (IF) and immunoblotting (IB): rat monoclonal anti-α-tubulin (IF, 1:800; IB, 1:4000; MCA78G, Bio-Rad), rabbit polyclonal anti-LAT1 (IB, 1:4000; #5347, Cell Signaling Technology), rabbit polyclonal anti-LAT1 (IF, 1:200; KE026, Trans Genic Inc), mouse monoclonal anti-phospho-Hisotone H3 (pS10) (IF, 1:400; #9706, Cell Signaling Technology), mouse monoclonal anti-HA-tag (IF, 1:500; IB, 1:1000; M180-3, Medical and Biological Laboratories), mouse monoclonal anti-γ-tubulin (IF, 1:500; GTU-88, MilliporeSigma), mouse monoclonal anti-NuMA (IF, 1:200; sc-365532, Santa Cruz Biotechnology), mouse monoclonal anti-CD98 (IB, 1:1000; sc-376815, Santa Cruz Biotechnology), sheep polyclonal anti-TGN46 (IF, 1:1000; AHP500GT, Bio-rad), mouse monoclonal anti-Calnxin (IF, 1:400; sc-46669, Santa Cruz Biotechnology), mouse monoclonal anti-Aurora A (IF, 1:400; #610938, BD Biosciences), rabbit monoclonal anti-phosho-Aurora A (pT288) (IF, 1:100; #30792, Cell Signaling Technology), rabbit monoclonal anti-GM130 (IF, 1:100; #12480, Cell Signaling Technology), and mouse monoclonal anti-cyclin B1 (IF, 1:50; sc-245, Santa Cruz Biotechnology) antibodies.

Techniques: Staining, Transfection, Control, Cell Culture, Fluorescence

Journal: PLoS ONE

Article Title: Tripolin A, a Novel Small-Molecule Inhibitor of Aurora A Kinase, Reveals New Regulation of HURP's Distribution on Microtubules

doi: 10.1371/journal.pone.0058485

Figure Lengend Snippet: ( A) Chemical structure of Tripolin A and Tripolin B. (B) Graph showing IC 50 values (in µM) of Tripolin A (red) and Tripolin B (green) in the presence of different ATP concentrations, using an in vitro kinase assay. (C) Differential Scanning Fluorimetry results for Aurora A in the presence and absence of the inhibitors. Blue curve determines the melting temperature of Aurora A alone (45°C), red in the presence of Tripolin A (47°C) and green in the presence of Tripolin B (53°C).

Article Snippet: Cells were blocked in PBS/5% w/v BSA pH 7.4 and stained with various combinations of: anti-Aurora A pT288 rabbit polyclonal antibody (1∶100; Cell Signaling Technology), anti-Aurora A mouse monoclonal antibody (1∶2000; Abcam), anti-AIM-1/Aurora B mouse monoclonal antibody (1∶500; BD Biosciences), anti-pHistoneH3 (Ser10) rabbit polyclonal antibody (1∶1000; Millipore), anti-pericentrin rabbit polyclonal antibody (1∶2000; abcam), anti-α-tubulin mouse monoclonal antibody clone GTU-88 (1∶500; Sigma-Aldrich), anti-α-tubulin mouse monoclonal antibody (1∶1000; Santa Cruz Biotechnology), rabbit polyclonal antisera against HURP and TPX2 , , for 1 h at room temperature.

Techniques: In Vitro, Kinase Assay

Journal: PLoS ONE

Article Title: Tripolin A, a Novel Small-Molecule Inhibitor of Aurora A Kinase, Reveals New Regulation of HURP's Distribution on Microtubules

doi: 10.1371/journal.pone.0058485

Figure Lengend Snippet: (A) Representative immunofluorescence images of HeLa cells in metaphase treated with solvent control (DMSO), 20 µM Tripolin A or Tripolin B for 5 h and 24 h. In the merged images Aurora A is pseudocolored red, pAurora T288 green, DNA blue. (Scale bars, 5 µm). (B) Fluorescence intensity (% percentage) of pAurora A T288 on centrosomes and total Aurora A on spindles were quantified in control metaphase cells or cells treated with Tripolin A or Tripolin B (n≥20 cells for each group, from at least two independent experiments). **: 0.0010.05; (Mann-Whitney test, two-tailed). Error bars represent SEM. (C) Western Blot analysis for Aurora A, Aurora B and pHistone H3 Ser10 in Tripolin A and Tripolin B-treated mitotic cells. α-tubulin was used as a loading control. (D) Representative immunofluorescence images of bipolar metaphase HeLa cells treated with solvent control (DMSO), 20 µM Tripolin A or Tripolin B for 24 h. In the merged images pHistone H3 Ser10 is pseudocolored red, Aurora B green, DNA blue. (Scale bars, 5 µm).

Article Snippet: Cells were blocked in PBS/5% w/v BSA pH 7.4 and stained with various combinations of: anti-Aurora A pT288 rabbit polyclonal antibody (1∶100; Cell Signaling Technology), anti-Aurora A mouse monoclonal antibody (1∶2000; Abcam), anti-AIM-1/Aurora B mouse monoclonal antibody (1∶500; BD Biosciences), anti-pHistoneH3 (Ser10) rabbit polyclonal antibody (1∶1000; Millipore), anti-pericentrin rabbit polyclonal antibody (1∶2000; abcam), anti-α-tubulin mouse monoclonal antibody clone GTU-88 (1∶500; Sigma-Aldrich), anti-α-tubulin mouse monoclonal antibody (1∶1000; Santa Cruz Biotechnology), rabbit polyclonal antisera against HURP and TPX2 , , for 1 h at room temperature.

Techniques: Immunofluorescence, Fluorescence, MANN-WHITNEY, Two Tailed Test, Western Blot

Journal: PLoS ONE

Article Title: Tripolin A, a Novel Small-Molecule Inhibitor of Aurora A Kinase, Reveals New Regulation of HURP's Distribution on Microtubules

doi: 10.1371/journal.pone.0058485

Figure Lengend Snippet: (A) Representative immunofluorescence images of mitotic HeLa cells treated with DMSO, 20 µM Tripolin A for 24 h, 100 nM MLN8237 for 24 h or Aurora A siRNAs. In the merged images α-tubulin is pseudocolored red, DNA blue. (Scale bars, 5 µm). (B) Graph showing the percentage of normal, multipolar, misaligned, disorganized and monopolar figures in control mitotic cells (DMSO or control siRNAs) and mitotic cells treated with Tripolin A, MLN8237 or Aurora A siRNA (n = 300 cells for each group, from three independent experiments). (C) Western Blot analysis for Aurora A levels in Aurora A siRNA treated cells. α-tubulin was used as a loading control. (D) Images of mitotic HeLa cells treated with DMSO, 20 µM Tripolin A for 5 h and 24 h or Aurora A siRNA. In the merged images Aurora A is pseudocolored red, pericentrin green, DNA blue. (Scale bar 5 µm). (E) Graph showing the percentage of mitotic cells with fragmented centrosomes (up), or acentrosomal poles (down) in control mitotic cells (DMSO or control siRNA) and mitotic cells treated with Tripolin A, or Aurora A siRNA (n = 150 cells for each group, from three independent experiments).

Article Snippet: Cells were blocked in PBS/5% w/v BSA pH 7.4 and stained with various combinations of: anti-Aurora A pT288 rabbit polyclonal antibody (1∶100; Cell Signaling Technology), anti-Aurora A mouse monoclonal antibody (1∶2000; Abcam), anti-AIM-1/Aurora B mouse monoclonal antibody (1∶500; BD Biosciences), anti-pHistoneH3 (Ser10) rabbit polyclonal antibody (1∶1000; Millipore), anti-pericentrin rabbit polyclonal antibody (1∶2000; abcam), anti-α-tubulin mouse monoclonal antibody clone GTU-88 (1∶500; Sigma-Aldrich), anti-α-tubulin mouse monoclonal antibody (1∶1000; Santa Cruz Biotechnology), rabbit polyclonal antisera against HURP and TPX2 , , for 1 h at room temperature.

Techniques: Immunofluorescence, Western Blot