Cells from ∼90% confluent 6-well plates were trypsinized, washed with phosphate-buffered saline (PBS), resuspended in 100 μl of ELB lysis buffer (250 mM NaCl, 0.1% NP-40, 50 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid [HEPES], pH 7, 5 mM EDTA) and 25 μl of 5× sample buffer, boiled for 10 min, and stored at −80°C. 30 μg of sample were run on 12% acrylamide gels and transferred to nitrocellulose. Primary and secondary antibodies were diluted in 5% milk in Tris-buffered saline plus 0.1% Tween 20. Primary antibody dilutions were as follows: Aurora B 1:100 (BD Transduction), pAurora A/B/C 1:100 (Cell Signaling), INCENP TSS 1:4000 (a kind gift of Dr. Michael Lampson), α-tubulin 1:250 (DM1a; Sigma-Aldrich), pH3 S10 1:100 (Millipore), α-FLAG 1:10,000 (Sigma A8592).
Aurora b
Manufactured by BD
The Aurora B is a high-performance laboratory instrument designed for precise and reliable analysis. It incorporates advanced technology to deliver accurate and reproducible results.
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Lab products found in correlation
α tubulin, by Merck Group (3 mentions)
Flag tag (flag), by Merck Group (2 mentions)
Aurora a, by BD (2 mentions)
α flag, by Merck Group (1 mentions)
P aurora a b c, by Cell Signaling Technology (1 mentions)
Ph3s10, by Merck Group (1 mentions)
Hisprobe hrp conjugate, by Thermo Fisher Scientific (1 mentions)
Cyclin b1, by BD (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
H3s10ph, by Cell Signaling Technology (1 mentions)
Gtx102170, by GeneTex (1 mentions)
Foxm1, by Santa Cruz Biotechnology (1 mentions)
Cenp t, by Fortrea (1 mentions)
Survivin, by Novus Biologicals (1 mentions)
Cenp b, by Abcam (1 mentions)
P21 sc 6246, by Santa Cruz Biotechnology (1 mentions)
Survivin, by GeneTex (1 mentions)
Nocodazole, by Merck Group (1 mentions)
Aurora b, by Abcam (1 mentions)
β tubulin tub 2.1, by Merck Group (1 mentions)
9 protocols using aurora b
1
Western Blot Analysis of Aurora Kinases
2
Immunoblotting and Immunofluorescence Protocol
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Antibodies against ACA (HCT-0100, ImmunoVision, 1:5000), Aurora B (611082, BD Transduction Laboratories, WB: 1:1000, IF: 1:250), CDH1 (CC43, Millipore, 1:500), Cyclin B1 (554177, BD Pharmingen, 1:5000), EGFP (sc-9996, Santa Cruz, 1:2000), Flag (F1804, Sigma-Aldrich, 1:1000), FOXM1 (sc-500, Santa Cruz, 1:500 for WB; GTX-102170, GeneTex, 1:250 for ChIP), GAPDH (2118, Cell Signaling, 1:5000), HA (home-made, WB: 1:5000), ubiquitin-HRP (AUB01, Cytoskeleton, 1:1000), H3S10ph (9706, Cell Signaling for IF, 1:1000; 06-570, Upstate-Merck, for WB, 1:1000), and RepoMan (HPA030049, Sigma, WB: 1:1000, IF: 1:300) were obtained from the indicated sources. For detection of His-tagged ubiquitin in Supplemental Figures S4G and 5C, the PVDF membrane was incubated with HisProbe-HRP Conjugate (15165, Thermo Scientific, 1: 2500). Secondary HRP–conjugated antibodies were purchased from Dako (Heverlee, Belgium). Secondary Alexa 488, 555, and 633 antibodies were obtained from Invitrogen (Carlsbad, CA).
3
Centromere Protein Immunofluorescence Assay
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Aurora B (at concentration 1:200) from BD Transduction Laboratories; INCENP (1:200) from Abcam (ab23956); Survivin (1:250) from Novus Biologicals (NB500-201 lot:AB-1); Borealin (1:500) from MBL 1D11 (m147-3 lot:012); CENP-B 1:600 from Abcam (ab25734) or 1:400 for (ab167361); CENP-C 1:150 from Hybridoma clone LX191 (from Don Cleveland, University of California, San Diego, San Diego, CA) or 1:500 for MBL International PD030; CENP-H (1:150; from Song-Tao Liu, University of Toledo, Toledo, OH); CENP-T (1:500) from Covance (Don Cleveland, University of California, San Diego, San Diego, CA); ACA (1:150) from Antibodies Incorporated 15–234; Sgo1 (1:200) from Abcam (ab58023); H3T3ph (1:50) from Cell Signaling (#9714); H2AT120ph (1:100) from Active Motif (39391); Bub1 (1:100) from Abcam (ab54893); Zw10 (1:200) from Abcam (ab21582); Hec1 (1:200) from Thermo Fisher (MA1-23308); and Pol II S2p (1:250) from Abcam (ab252855).
4
Western Blot Analysis of Cell Signaling
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Western blot analysis was performed as we previously described [15 ]. The used antibodies are listed below. FOXM1 (sc-376471) and p21 (sc-6246) were purchased from Santa Cruz. Aurora-A (610404), Aurora-B (611083), BubR1 (612502) were purchased from BD Biosciences. Survivin (GTX100052) was purchased from GENETEX. Actin (A2228) was purchased from Sigma-Aldrich.
5
Antibody Profiling of Cell Cycle Regulators
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The following antibodies were used for immunoprecipitation (IP) and immunoblotting (IB, dilutions shown): NudC (70/1, rabbit, 1:1000) [32 (link)], NudC (G1, goat, 1:1000) [33 (link)], NudC (2D9, mouse, 1:2000) [32 (link)], Aurora B (BD, mouse, 1:800), Aurora B (Abcam, rabbit, 1:2000), α-tubulin (GeneTex, rabbit, 1:2000), α-tubulin (Sigma, mouse, 1:1000), β-tubulin(tub2.1) (Sigma, mouse, 1:1000), and FLAG (Sigma, mouse, 1 μl/mg protein for IP). The following antibodies were used for immunofluorescence (1:1000, unless otherwise indicated): NudC (G1, goat) [33 (link)], pSerNudC (R2, rabbit) [32 (link)], Borealin (MBL International, mouse), PRC1 (Abcam, rabbit), MKLP-1 (Cell Signaling, rabbit), and pTSS-INCENP834-902 (gift of Dr. Michael Lampson, University of Pennsylvania; rabbit) [36 (link)], Spc25 (gift of Dr. P. Todd Stukenberg, University of Virginia Medical Center; rabbit; 1:700) [37 (link)], and CREST-SH autoserum (gift of Dr. Bill R. Brinkley, Baylor College of Medicine; human; 1:10,000) [38 (link)]. Protease inhibitors and nocodazole, the microtubule depolymerizing agent, were purchased from Sigma. ZM447439, an Aurora B inhibitor, was purchased from Tocaris and used at a final concentration of 2 μM.
6
Immunofluorescent Staining of Murine Mammary Glands
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Mammary glands were harvested from 8- to 9-week-old female mice and prepared and stained according to published records (Jechlinger et al., 2009 (link)). The following primary antibodies were used: HA (1:,1000, Covance, MMS-101R), cleaved Caspase3 (1:200, Cell Signaling Technology, 9661S), LaminB1 (1:500, Abcam, 16048), α-Tubulin (1:500, Sigma, F2168), and Aurora B (1:150, BD Biosciences, 611082). Imaging of fixed samples was performed on a Leica TCS SP5/SP8 confocal microscope and time-lapse imaging during 20 hr on an inverted spinning disk confocal microscope (PerkinElmer Ultraview-Vox): 0.3-μm optical sectioning across a 35-μm stack, 5 frames/hr. Volocity version 6.2 (Improvision, PerkinElmer) served for image acquisition and analysis.
7
Western Blot Analysis of Cell Signaling
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Essentially, cell lysates were prepared in RIPA cell lysis buffer containing protease and phosphatase inhibitor cocktails (Roche); samples were resolved in 10% SDS-PAGE gel and transferred to a nitrocellulose membrane (Millipore). The membrane was blocked for 1 hr in 3% BSA solution at room temperature, followed by incubation with primary antibodies overnight at 4°C at indicated dilutions (mouse monoclonal Aurora A (BD Biosciences 610938) 1 : 1000, Aurora B (BD Biosciences 611082) 1 : 1000, Plk1 (Abcam ab17056) 1 : 2000, Cdk1 (Abcam ab32384) 1 : 5000, rabbit polyclonal total Elk-1 (Cell Signaling #9182) 1 : 1000, rabbit P-S383-Elk-1 (Cell Signaling #9181) 1 : 1000, rabbit p44/42 MAPK (Erk1/2) (Cell Signaling #9102) 1 : 1000, rabbit SRF (Santa Cruz Biotechnology sc-335) 1 : 1000, and rabbit GST (Cell Signaling #2622) 1 : 1000). Blots were then washed 3 times in TBS-T and incubated with secondary antibodies (goat anti-mouse IgG-HRP, Santa Cruz sc-2005 or anti-rabbit IgG- HRP, Santa Cruz sc-2004) at 1 : 5000 dilution in BSA for 1 hr. After 3 washes in TBS-T, signal was developed with Immobilon Crescendo Western HRP substrate (Millipore) and captured with either Bio-Rad or DNS chemiluminescent imaging systems.
8
Characterization of Mitotic Spindle Proteins
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Standard lab reagents and stocks were purchased from Sigma Aldrich, Thermo Rabbit antibodies against PLK1, PRC1 and PRC1-pT602 were described previously (Neef et al., 2007; Neef et al., 2006) . The MAD2 antibody was raised in sheep (Scottish Blood Transfusion Service) using full length human 6His-MAD2 expressed in bacteria and coupled to KLH as the antigen. The serum was affinitypurified using GST-tagged MAD2. The commercial antibodies used were Aurora A (Cell Signalling; 4718S), Aurora B (BD Transduction labs; 611083), BUBR1 (Bethyl; A300-386A), CDC20 (Proteintech; 10252-1), CDH1 (Santa Cruz; sc-56312), Cyclin B1 (Millipore; 05-373), PPP1CA (Bethyl; A300-904A), PPP1CA-pT320 (AbCam, ab62334), PRC1-pT481 (AbCam; EP1514Y/ab62366), securin (AbCam; ab79546), TPX2 (AbCam; ab32795), tubulin (Sigma; T6199). For Western blotting, proteins were separated by SDS-PAGE and transferred to nitrocellulose using a Trans-blot Turbo system (Bio-Rad Laboratories). Species-specific secondary antibodies conjugated to horseradish peroxidase (HRP) were obtained from Jackson ImmunoResearch Laboratories, Inc. and used at 1 µg/ml. All Western blots were revealed using ECL (GE Healthcare) on films.
9
Comprehensive Immunohistochemistry and Western Blot Protocol
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