HeLa cells were plated on poly-L-lysine coated circular coverslips (0.1mg/ml poly-L-lysine) and allowed to adhere overnight prior to transfection with control or AURKB-targeting siRNA (siAURKB, Dharmacon). Cells were treated with 1 µM S-trityl-L-cysteine (STLC), a specific inhibitor of human mitotic kinesin Eg5 [31 ,32 ], for 18 hr to enrich the mitotic population. HeLa cells were also treated with STLC in combination with MG132 and hesperadin to enrich the mitotic population and inhibit the kinase activity of AURKB. The cells were subsequently subjected to co-staining with anti-P-S92-HP1α (1:50, Abcam) and anti-Aurora B (1:50, Abcam) or anti-INCENP (1:200, Thermo Fisher Scientific). Proximity ligation was performed using the DuoLink® PLA kit according to the manufacturer’s instructions (Sigma-Aldrich). The PLA experiments are quantified by counting the number of dots per cell in 150 cells, n = 3 independent experiments. Statistical significance was assessed with Student’s t-test in GraphPad Prism 7.
Anti aurora b
Manufactured by Abcam
Sourced in United States, United Kingdom
Anti-Aurora B is a primary antibody that binds to and detects the Aurora B protein, which is a serine/threonine protein kinase involved in regulating cell division. This antibody can be used in various immunoassay techniques to study the expression and localization of Aurora B in biological samples.
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Lab products found in correlation
Anti ki67, by Abcam (6 mentions)
Anti α actinin, by Merck Group (4 mentions)
4 6 diamidino 2 phenylindole (dapi), by Merck Group (3 mentions)
Anti brdu, by Merck Group (2 mentions)
Anti ctnt, by Abcam (2 mentions)
Axioscan, by Zeiss (2 mentions)
Anti ptie2 tyr992, by Merck Group (2 mentions)
Anti angpt4, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 647 goat anti chicken igy h l, by Abcam (2 mentions)
Anti egfp, by Abcam (2 mentions)
Anti mef2c, by Abcam (2 mentions)
Anti p erk, by Cell Signaling Technology (2 mentions)
Alexa fluor 594 goat anti rabbit igg, by Thermo Fisher Scientific (2 mentions)
Alexa fluor 488 goat anti mouse igg, by Thermo Fisher Scientific (2 mentions)
Anti cd31, by Abcam (2 mentions)
Gapdh, by Cell Signaling Technology (2 mentions)
Mouse monoclonal anti α tubulin clone dm1a, by Merck Group (2 mentions)
Peroxidase and, by Jackson ImmunoResearch (2 mentions)
Mouse monoclonal anti flag clone m2, by Merck Group (2 mentions)
Alexa fluor conjugated secondary antibody, by Jackson ImmunoResearch (2 mentions)
17 protocols using anti aurora b
1
Proximity Ligation Assay for Aurora B
2
Immunofluorescence Staining of Cell Markers
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Cells were rinsed in PBS, fixed for 10 min with 4% formaldehyde in PBS and permeabilized 5 min with 0.1% Triton-X100. After fixation, coverslips were blocked in PBS containing 3% bovine serum albumin for 30 min at 37°C, before being processed for immunofluorescence.
Ki67 immunostaining on tissue slices has been performed following standard procedures.
Antibody dilution was as follows: anti-RhoA-GTP (NewEast Biosciences) 1:400; anti-Aurora B (AbCam ab3609) 1:100; anti-E-Cadherin (BD Biosciences Pharmingen, Bedford, MA) 1:50; anti-Snail (AbCam) 1:50; anti-Ki67 (BD Bioscience Pharmingen, USA; 1:25)
Secondary antibodies conjugated to Alexa-488 or Alexa-594 (Molecular Probes, Invitrogen, San Diego, CA; 1:400) were used as recommended by the supplier. Alexa Fluor 594 Phalloidin (a12381) (Thermo Fisher Scientific Inc., MA, USA) and Alexa Fluor 647–Phalloidin (from Invitrogen, Carlsbad, CA) were utilized for the staining of F-actin.
DNA was counterstained with 0.1 μg/ml 4'-6'-Diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St Louis, MO).
All stained samples were analyzed under a Nikon Microphot-FXA microscope equipped with a CCD camera and digital images were acquired with Nikon NIS-elements software or using a Leica SP5 spectral confocal microscope.
Ki67 immunostaining on tissue slices has been performed following standard procedures.
Antibody dilution was as follows: anti-RhoA-GTP (NewEast Biosciences) 1:400; anti-Aurora B (AbCam ab3609) 1:100; anti-E-Cadherin (BD Biosciences Pharmingen, Bedford, MA) 1:50; anti-Snail (AbCam) 1:50; anti-Ki67 (BD Bioscience Pharmingen, USA; 1:25)
Secondary antibodies conjugated to Alexa-488 or Alexa-594 (Molecular Probes, Invitrogen, San Diego, CA; 1:400) were used as recommended by the supplier. Alexa Fluor 594 Phalloidin (a12381) (Thermo Fisher Scientific Inc., MA, USA) and Alexa Fluor 647–Phalloidin (from Invitrogen, Carlsbad, CA) were utilized for the staining of F-actin.
DNA was counterstained with 0.1 μg/ml 4'-6'-Diamidino-2-phenylindole (DAPI, Sigma-Aldrich, St Louis, MO).
All stained samples were analyzed under a Nikon Microphot-FXA microscope equipped with a CCD camera and digital images were acquired with Nikon NIS-elements software or using a Leica SP5 spectral confocal microscope.
3
Cardiac Cell Proliferation Analysis
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The frozen sections of the heart (4 μm) from the P0 and P7 rats or the cells were fixed in 4% paraformaldehyde solution at room temperature for 12 min and washed three times with PBS. Then 10% normal goat serum and 5% bovine serum albumin in 1X PBS was used to block the sections or the cells for 1 h at room temperature. The sections were incubated overnight at 4°C with primary antibodies as follows: anti-Ki67 (Abcam, ab16667, 1:200), anti-α-Actinin (Cell Signaling Technology, Danvers, MA, USA, 69758, 1:200), anti-Aurora B (Abcam, ab2254, 1:200), and anti-pH3 (Cell Signaling Technology, Danvers, MA, USA, 69758, 1:500). After washing three times with PBS, the sections were incubated with Alexa-488- or Alexa-cy3-conjugated secondary antibodies at room temperature for 1 h followed by washing three times with PBS. Finally, microscopic images were obtained using a fluorescent microscope (Carl Zeiss, Oberkochen, Germany). The positive percentages of Ki67, Aurora B, and pH3 were quantified using NIH Image J software (MD, USA).
4
Immunofluorescence Staining of Cardiac Cells
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Cells were fixed with 4% paraformaldehyde for 15 min and permeabilized with 0.5% Triton X-100 (Sigma) in 1× PBS for 10 min at room temperature. After 1× PBS washing, 4% FBS was used for blocking for 1 h at room temperature. Then, cells were incubated with the primary antibodies (1:10 to 1:100 dilution), including anti-α-actinin (Sigma, A7811), anti-Ki67 (Abcam, ab15580), anti-Aurora B (Abcam, ab2254) or anti-cTnT (sc-20025, Santa Cruz) overnight at 4°C, and the secondary antibody conjugated to Alexa Fluor 488 (Invitrogen, A11001) or Alexa Fluor 555 (Invitrogen, A21428) for 1 h at room temperature. The nucleus was stained with 4′,6-diamidino-2-phenylindole (DAPI, Sigma, D9542) for 30 min at room temperature. For EdU staining, the culture medium containing 10 μM EdU (Invitrogen, C10339) was applied to H9C2 or NRVM cells for 2 or 24 h, respectively, followed by EdU analysis using Click-iT EdU imaging kits (Invitrogen). All slides were imaged using fluorescence microscopy (Leica, Germany).
5
Immunofluorescence Staining of Cell Markers
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Deparaffinization, antigen retrieval with 1 mM EDTA (pH 9.0) in boiling water, and blocking of nonspecific binding sites were performed. The sections were then incubated with primary antibodies overnight at 4°C, washed three times with PBS, and incubated with fluorescence-labeled secondary antibodies for 1 h at 25°C in the dark. The slides were washed three times in PBS, counterstained with DAPI (Sigma-Aldrich, St. Louis, MO, USA), and mounted with VECTASHIELD (Vector Labs, CA, USA). The primary antibodies used were as follows: anti-phospho Histone H3 Ser10 (Millipore #06-570, 1 : 100), anti-Ki67 (Abcam, ab16667, 1 : 200), anti-Aurora B (1 : 100; ab2254, Abcam), and anti-Sarcomeric Alpha Actinin (Abcam, ab9465, 1 : 500). The anti-rabbit Alexa Fluor 488-conjugated (1 : 500; A-21206) and anti-mouse Alexa Fluor 594-conjugated (1 : 500; A-21203) secondary antibodies were from Invitrogen. Fluorescence was observed under a ZEISS LSM800 confocal laser scanning microscope (Carl Zeiss, Inc., Jena, Germany).
6
Immunofluorescence Staining of Cardiac Markers
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Immunofluorescence staining was performed on cryosections as previously described (Han et al., 2014 (link)). The primary antibodies used in this study were: anti-EGFP (Abcam), anti-pERK (Cell Signaling Technology), anti-pTie2 (Tyr992) (Millipore), anti-Mef2c (Abcam), anti-MF20 (DSHB), anti-BrdU (Sigma), anti-α-actinin (Sigma), anti-CD31(Abcam), anti-Angpt4 (Invitrogen), anti cTnT (Abcam), anti-Ki67 (Abcam), and anti-Aurora B (Abcam). The secondary antibodies used in this study were: Alexa Fluor 488 goat anti-mouse IgG (Invitrogen), Alexa Fluor 594 goat anti-rabbit IgG (Invitrogen), Alexa Fluor 647 goat anti-chicken IgY H&L (Abcam). Nikon A1 confocal microscope and Zeiss Axio Scan were used to observe and record the immunostaining images. To quantify the pERK and pTie2 signals, heart area and pERK/pTie2 positive area were analyzed using Surface function in Imaris, then we calculated the ratio of pERK/pTie2 positive area/heart section area.
7
Immunostaining of Cardiac Cells
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The NRVMs were fixed with 4% paraformaldehyde for 10 min. The fixed cells and tissue sections were treated with Triton (0.1%) for 8 min and blocked with blocking buffer (Solarbio) for 30 min at 37°C. Then, the samples were incubated with the primary antibodies overnight at 4°C. After washing by PBS, the samples were incubated with fluorescent secondary antibodies for 1 h at 37°C, followed by 5 min of DAPI staining. After staining, microscopy was performed using Olympus confocal microscope (FluoView 1000). The primary antibodies used were anti-cardiac troponin T (#MA5-12960, Thermo Fisher Scientific), anti-α actinin (#A7811, Sigma), anti-Ki67 (#9027S, Cell-Signaling Technology), anti-pH3 (#PA5-17869, Thermo Fisher Scientific), and anti-Aurora B (#ab2254, Abcam). The secondary antibodies used were goat anti-mouse IgG (H+L), Alexa Fluor Plus 488 (#A-32723, Thermo Fisher Scientific), Goat anti-rabbit IgG (H+L), and Alexa Fluor Plus 488 (#A-11035, Thermo Fisher).
8
Indirect Immunostaining of Cytoskeletal and Chromosomal Proteins
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The following primary antibodies were used for indirect immunostaining: mouse monoclonal anti-α-tubulin (1:1,000; Calbiochem), mouse monoclonal anti-acetylated tubulin (1:1,000; Sigma-aldrich), rabbit polyclonal anti-CENP-B (1:1,000; Millipore), rabbit polyclonal anti-histone H3K9me3 (1:2,500; Millipore), mouse monoclonal anti-HP1α (1:400; Millipore), rabbit polyclonal anti-Aurora B (1:500; Abcam), mouse monoclonal anti-HEC1 (1:200; Abcam), rabbit polyclonal anti-Mis12 (1:200; Abcam), mouse monoclonal anti-CENP-F (1:200; Abcam), and human anti-centromere autoantibody (CREST, 1:1,000; Cortex Biochem).
9
Cell Cycle Regulation Analysis Protocol
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Thymidine, nocodazole, propidium iodide (PI) and antibodies against β-actin and Flag were purchased from Sigma. Doxycycline was obtained from Clontech. CRYSTAL VIOLET was purchased from Amresco. RO-3306 was purchased from Calbiochem. ProLong Gold antifade reagent was purchased from Life Technology. Antibody against CREPT (3E10) was produced in this lab26 (link). Anti-tubulin antibody was purchased from CMCTAG. Anti-Myc (9E10), anti-HA (F-7), anti-Cyclin B1(H-433), anti-Cyclin A (C-19), and anti-Cyclin E (HE12) antibodies were purchased from Santa Cruz Biotechnology. Antibodies against histone H3 and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were purchased from Cell Signaling Technology. Anti-Aurora B, anti-Cyclin D1, and anti-Cyclin B1 (ab32053) antibodies were purchased from Abcam. Anti-H3S10p antibody was purchased from Millipore. Fluorescent secondary antibodies (goat anti-rabbit IgG and goat anti-mouse IgG) were purchased from Jackson ImmunoReseach.
10
Immunohistochemical Analysis of Aurora-B and pNPM1 in Osteosarcoma
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A total of 87 tissue sections histologically diagnosed with OS were obtained from the First Affiliated Hospital of Nanchang University, China. No patients received therapy prior to biopsy. All experiments were approved by the ethics committee of the First Affiliated Hospital of Nanchang University (Jiangxi, China; NO.Y2019-126) and followed the Declaration of Helsinki. All the subjects were informed of the contents, latent risks, objectives and signed written informed consents.
Immunoperoxidase procedures (Maxvasion procedure) were performed to detect relative protein expression. Briefly, sections were heated in citrate buffer for 20 min and probed with anti-Aurora-B (1:500, Abcam, MA, USA) and pNPM1Ser125 (1:1000, Abcam) antibodies overnight. Stained sections were scored by two doctors, respectively. Tissues showing yellow and brown nuclear staining were used to indicate a positive result, and tissues lacking staining in the nucleolus were considered negative. Immunohistochemical expression of Aurora-B and pNPM1Ser125 were determined as “-”, “+”, “++”, and “+++” according to the coloring intensity and the percentage of positively stained tumor cells.
Immunoperoxidase procedures (Maxvasion procedure) were performed to detect relative protein expression. Briefly, sections were heated in citrate buffer for 20 min and probed with anti-Aurora-B (1:500, Abcam, MA, USA) and pNPM1Ser125 (1:1000, Abcam) antibodies overnight. Stained sections were scored by two doctors, respectively. Tissues showing yellow and brown nuclear staining were used to indicate a positive result, and tissues lacking staining in the nucleolus were considered negative. Immunohistochemical expression of Aurora-B and pNPM1Ser125 were determined as “-”, “+”, “++”, and “+++” according to the coloring intensity and the percentage of positively stained tumor cells.
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