Aurka

Manufactured by Cell Signaling Technology

Sourced in United States

AURKA is a laboratory product manufactured by Cell Signaling Technology. It is an enzyme that plays a crucial role in the regulation of cell division. The core function of AURKA is to facilitate the proper assembly and function of the mitotic spindle during cell division.

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Lab products found in correlation

Close spelling variants of this product

Anti-AURKA (10 citations) P-AURKA (5 citations) Phospho-AURKA (4 citations) P-AURKA (Thr288) (4 citations) Aurora kinase A (AURKA) (2 citations) Rabbit anti-AURKA (2 citations)

26 protocols using aurka

Comprehensive Protein Expression Analysis

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Antibodies against GAPDH (ET1601‐4, Huabio), AURKA (14475S, Cell Signaling Technology), PARP (9532S, Cell Signaling Technology), c‐Myc (AF6513, Beyotime), pH3S10 (9701S, Cell Signaling Technology), H3 (4499, Cell Signaling Technology), Bcl‐2 (15071, Cell Signaling Technology), CRBN (AF6564, Beyotime), cIAP1 (GTX110087, GeneTex), VHL (68547, CST), cyclin E1 (AF2491, Beyotime), cyclin B1 (AF1606, Beyotime), STAT5A (AF2038, Beyotime), NANOG (ab203919, Abcam), FOXM1 (AF6924, Beyotime), AURKB (GTX132702, GeneTex), TACC3 (AF1345, Beyotime), TPX2 (GTX115654, GeneTex), Phospho‐TACC3 (Ser558) (AF4506, Affinity Biosciences), and Phospho‐Aurora A (Thr288)/Aurora B (Thr232)/Aurora C (Thr198) (2914S, CST) were used for the Western blot analysis. PE antihuman CD34 (343506, Biolegend) and Alexa Fluor 700 antimouse/human CD11b (101222, Biolegend) were used for the flow cytometry analysis. AURKA (14475S, Cell Signaling Technology), α‐Tubulin (AT819, Beyotime), Alexa Fluor 488 goat antirabbit IgG (H+L) (A11034, Invitrogen), Alexa Fluor 546 goat antimouse IgG (H+L) (A11003, Invitrogen) were used for immunofluorescence assay.

Liu F., Wang X., Duan J., Hou Z., Wu Z., Liu L., Lei H., Huang D., Ren Y., Wang Y., Li X., Zhuo J., Zhang Z., He B., Yan M., Yuan H., Zhang L., Yan J., Wen S., Wang Z, & Liu Q. (2022). A Temporal PROTAC Cocktail‐Mediated Sequential Degradation of AURKA Abrogates Acute Myeloid Leukemia Stem Cells. Advanced Science, 9(22), 2104823.

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Alisertib-Mediated Signaling Pathway Study

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Alisertib was prepared and stored according to the manufacturer’s instructions (Millennium Pharmaceuticals, Inc., Cambridge, MA). Specific antibodies against p-MTOR(Ser2448), p-MEK1/2(Ser217/221), p-AKT(Ser473), p-AURKA(T288), mTOR, MEK, AKT, AURKA, RPS6KB1, p-RPS6KB1(T389), Phospho-(Ser/Thr) Phe, and β-Actin were purchased from Cell Signaling Technology (Beverly, MA). Recombinant human AURKA and RPS6KB1 proteins were obtained from Cell Sciences (Canton, MA). Specific antibodies against KRAS were purchased from Santa Cruz Biotechnology (Dallas, TX). KRAS-G12D lentiviral vector was purchased from Applied Biological Materials (Richmond, BC, Canada). Tet-One™ Inducible Expression System and Tet System Approved FBS were purchased from Clontech (Palo Alto, CA). Transfection reagent LipoJet was purchased from SignaGen Laboratories (Gaithersburg, MD). Tet-on expression system, doxycycline-free fetal bovine serum, and human cell cycle arrays were purchased from Clontech (Palo Alto, CA).

Wang-Bishop L., Chen Z., Gomaa A., Lockhart A.C., Salaria S., Wang J., Lewis K.B., Ecsedy J., Washington K., Beauchamp R.D, & El-Rifai W. (2018). Inhibition of AURKA Reduces Proliferation and Survival of Gastrointestinal Cancer Cells With Activated KRAS by Preventing Activation of RPS6KB1. Gastroenterology, 156(3), 662-675.e7.

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Transfection of Apoptosis-Related Genes

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Transfections were performed in A375 and Hs294T cells using Lipofectamine RNAi Max (Thermofisher) according to the manufacturer’s protocol. We used siRNA targeting p53 (Santa Cruz Biotechnology), BAX (Cell Signaling), PUMA (Cell Signaling), AURKA (Cell Signaling), p21 (Cell Signaling), Bak (Cell Signaling), BCL-xL (Thermo Fisher), and control non-targeting siRNA (Cell Signaling).

Bharti V., Watkins R., Kumar A., Shattuck-Brandt R.L., Mossing A., Mittra A., Shen C., Tsung A., Davies A., Hanel W., Reneau J.C., Chung C., Sizemore G.M., Richmond A., Weiss V.L, & Vilgelm A.E. (2022). BCL-xL inhibition potentiates cancer therapies by redirecting the outcome of p53 activation from senescence to apoptosis. Cell reports, 41(12), 111826.

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Western Blot Analysis of Cell Cycle Regulators

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Western blots were conducted as previously described [25 (link)]. Primary antibodies specific to CDK1, CCNB1, CCNE2, BIRC5, AURKA, FOXM1, WDR5, H3K4me3 (1:1000, Cell Signalling Technology, Danvers, MA, USA), histone H3 (1:2000, Cell Signalling Technology, Danvers, MA, USA), XRCC2, MCM2, PLK1, and GAPDH (1:1000, Abcam) were used. The PVDF films were then incubated with secondary antibodies (anti-mouse or anti-rabbit, Promega, USA) and visualized using Immobilon enhanced chemiluminescence (Millipore). The full images of all Western blots are shown in Supplementary Fig. 9.

Zhang J., Zhou Q., Xie K., Cheng L., Peng S., Xie R., Liu L., Zhang Y., Dong W., Han J., Huang M., Chen Y., Lin T., Huang J, & Chen X. (2021). Targeting WD repeat domain 5 enhances chemosensitivity and inhibits proliferation and programmed death-ligand 1 expression in bladder cancer. Journal of Experimental & Clinical Cancer Research : CR, 40, 203.

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Antibody Panel for Cell Signaling Pathways

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The antibodies to c-PARP (#9544), p-MLKL (Ser358,
#91689), MLKL (human specific, #14993), MLKL (mouse specific,
#28640), p-H3 (Ser10, #3377), GSK3β (#9315),
p-GSK3β (Ser9, #9336), RIPK3 (#13526),
p-AURKA(Thr288)/AURKB(Thr232)/AURKC(Thr198) (#2914), AURKA (human
specific, #14475), Flag (#14793), and HA (#3724) were
purchased from Cell Signaling Technology. The antibodies to GAPDH
(#G8795) and β-actin (#A5441) were purchased from Sigma
Aldrich. The antibodies to RIPK1 (#NB100-56160), LC3
(#NB100-2220), HMGB1 (#H00003146-M08), and AURKA
(#NBP2-36737) were purchased from Novus Biologicals. The antibodies to
GPX4 (#ab125066) and p-MLKL (Ser345, # ab196436) were purchased
from Abcam. The antibody to p-AURKA (Thr288, # GTX55002) was purchased
from GeneTex Inc. The antibody to TNFR1 (#MAB225R) was purchased from
R&D Systems. CCT137690 (#S2744), ferrostatin-1 (#S7243),
AR-A014418 (#S7435), staurosporine (#S1421), erastin
(#S7242), and kinase inhibitor library (#L1200) were obtained
from Selleck Chemicals. Necrostatin-1 (#N9037) and gemcitabine
(#G6423) were purchased from Sigma Aldrich. Z-VAD-FMK (#219007)
and XXVI (#361569) were purchased from EMD Millipore. Chloroquine
(#193919) was purchased from MP Biomedicals. Necrosulfonamide
(#5025) and recombinant human TNF (#210-TA-005) were purchased
from R&D Systems.

Xie Y., Zhu S., Zhong M., Yang M., Sun X., Liu J., Kroemer G., Lotze M., Zeh HJ I.I.I., Kang R, & Tang D. (2017). Inhibition of Aurora Kinase A Induces Necroptosis in Pancreatic Carcinoma. Gastroenterology, 153(5), 1429-1443.e5.

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Immunoblot and Immunofluorescence Assay Protocol

Cited 1 time

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Immunoblot and immunofluorescence assays were performed as previously described [29 (link)]. Antibodies employed to perform these studies were as follows: centrin (20H5 kindly provided by Dr. Salisbury’s laboratory at the Mayo Clinic); ERα and pericentrin (Santa Cruz Biotechnology, Dallas, TX, USA); AURKA (Cell Signaling Technology, Danvers, MA, USA); CD44, CD24, NOTCH1, NOTCH2, and NOTCH3 (Abcam, Cambridge, MA, USA); and β-actin and α-tubulin (Sigma-Aldrich, St. Louis, MO, USA). Secondary antibodies were obtained from Molecular Probes (Eugene, OR, USA).

Leontovich A.A., Jalalirad M., Salisbury J.L., Mills L., Haddox C., Schroeder M., Tuma A., Guicciardi M.E., Zammataro L., Gambino M.W., Amato A., Di Leonardo A., McCubrey J., Lange C.A., Liu M., Haddad T., Goetz M., Boughey J., Sarkaria J., Wang L., Ingle J.N., Galanis E, & D’Assoro A.B. (2018). NOTCH3 expression is linked to breast cancer seeding and distant metastasis. Breast Cancer Research : BCR, 20, 105.

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Histone H3 and Cell Signaling Protein Analysis

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Membranes were blotted with antibodies directed against Ser-10 phosphorylated histone H3 (1:500) and GAPDH (1:5000) from Millipore, histone H3 (1:1000), c-ABL (1:1000), FLT3 (1:1000), AURKA (1:1000), AURKB (1:1000) from Cell Signaling, AURKC (1:200, Zymed), and β-tubulin (1:3000, Calbiochem). Bound antibodies were detected with horseradish peroxidase–linked antibodies against mouse or rabbit immunoglobulin G (Amersham), followed by ECL (Amersham).

Li N., Maly D.J., Chanthery Y.H., Sirkis D.W., Nakamura J.L., Berger M.S., James C.D., Shokat K.M., Weiss W.A, & Persson A.I. (2014). Radiotherapy Followed by Aurora Kinase Inhibition Target Tumor-Propagating Cells in Human Glioblastoma. Molecular cancer therapeutics, 14(2), 419-428.

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Immunoblotting of Whole-Cell Protein Extracts

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Whole-cell protein extracts were prepared with ice-cold RIPA buffer (25 mM Tris-HCl pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)) with Complete Protease Inhibitor Cocktail (Roche Life Sciences, Indianapolis, IN). Each aliquot of protein sample was run on a SDS-polyacrylamide gel electrophoresis and transferred onto a nitrocellulose membrane for immunoblotting with primary antibodies, including ARID1A (Santa Cruz, sc-32761, 1:1000 dilution), CDC25C (Santa Cruz, sc-327, 1:2000 dilution), CDC2 (Santa Cruz, sc-54, 1:2000 dilution), GAPDH (Santa Cruz, sc-365062, 1:5000 dilution), HSP90 (Santa Cruz, sc-69703, 1:5000 dilution), α-tubulin (Santa Cruz, sc-5286, 1:5000 dilution), p-CDC25C (Cell Signaling, #9529s, 1:1000 dilution), p-CDC2 (Cell Signaling, #9111s, 1:1000 dilution), AURKA (Cell Signaling, #14475s, 1:2000 dilution), and cleaved caspase 3 (Cell Signaling, #9661s, 1:2000 dilution) antibodies, followed by horseradish peroxidase-conjugated secondary antibodies (Santa Cruz). Uncropped versions of all blots are shown in Supplementary Figs. 8-12.

Wu C., Lyu J., Yang E.J., Liu Y., Zhang B, & Shim J.S. (2018). Targeting AURKA-CDC25C axis to induce synthetic lethality in ARID1A-deficient colorectal cancer cells. Nature Communications, 9, 3212.

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Comprehensive Protein Analysis Workflow

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Proteins were extracted using RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM
NaCl, 0.1% sodium deoxycholate, 0.1% SDS, 1 mM EDTA pH 8.0,
1% NP-40) containing proteinase (Roche) and phosphatase (Roche)
inhibitor cocktails. Samples were resolved using 4–12% SDS-PAGE
gels (Life Technologies) and transferred to PVDF membranes (Millipore).
Membranes were probed overnight on a 4°C shaker with primary antibodies
(1:1,000 dilution unless indicated) recognizing the following proteins: p-AKT
(Ser473) (9271, Cell Signaling), AKT (4691, Cell Signaling), p-S6 (Ser240/244)
(5364, Cell Signaling, 1:20,000), p-4E-BP1 (Thr37/46) (2855, Cell Signaling),
p-AURKA (Thr288) (3079, Cell Signaling), AURKA (4718, Cell Signaling), Cleaved
PARP (Asp214) (9541, Cell Signaling), BCL2 (2870, Cell Signaling), BAX (2772,
Cell Signaling), MYC (ab32072, Abcam), and β-actin (3700, Cell
Signaling).

Donnella H.J., Webber J.T., Levin R.S., Camarda R., Momcilovic O., Bayani N., Shah K.N., Korkola J., Shokat K.M., Goga A., Gordan J.D, & Bandyopadhyay S. (2018). Kinome rewiring reveals AURKA limits PI3K-pathway inhibitor efficacy in breast cancer. Nature chemical biology, 14(8), 768-777.

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Immunohistochemical Analysis of Mouse Tissues

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Western blot analysis of mouse tissues and neurospheres was performed as previously described (Brockmann et al., 2013; Chesler et al., 2006 ). For IHC of mouse tissues, samples were fixed in 4% paraformaldehyde in phosphate buffered saline for at least 24 hr, decalcified with 0.3 M EDTA, and processed using a Leica ASP300S tissue processor. Sections were cut at 4 μM for hematoxylin and eosin staining (H&E) staining and immunohistochemistry as previously described (Chesler et al., 2006 (link)). Antibodies used were MYCN (OP-13, Merck-Millipore), Ki-67 (556003, BD Biosciences), GFAP (Z0334, DAKO), Cleaved Caspase 3 (9664, Cell Signaling Technology), Synaptophysin (180130, Life Technologies), Phospo-S10-Histone H3 (9706, Cell Signaling), phospho-AurkABC (2914, Cell Signaling), AurkA (4718, Cell Signaling), Sonic Hedgehog (ab73958, Abcam), Gli-1 (2534, Cell Signaling), and GAPDH (2118, Cell Signaling).

Hill R.M., Kuijper S., Lindsey J.C., Petrie K., Schwalbe E.C., Barker K., Boult J.K., Williamson D., Ahmad Z., Hallsworth A., Ryan S.L., Poon E., Robinson S.P., Ruddle R., Raynaud F.I., Howell L., Kwok C., Joshi A., Nicholson S.L., Crosier S., Ellison D.W., Wharton S.B., Robson K., Michalski A., Hargrave D., Jacques T.S., Pizer B., Bailey S., Swartling F.J., Weiss W.A., Chesler L, & Clifford S.C. (2015). Combined MYC and P53 Defects Emerge at Medulloblastoma Relapse and Define Rapidly Progressive, Therapeutically Targetable Disease. Cancer Cell, 27(1), 72-84.

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