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).
P aurora a b c
Manufactured by Cell Signaling Technology
Sourced in United States
The P-Aurora A/B/C product is an antibody that specifically detects the phosphorylated forms of Aurora A, Aurora B, and Aurora C kinases. These kinases play crucial roles in regulating cell division and are important targets in cancer research.
Automatically generated - may contain errors
Lab products found in correlation
Cleaved caspase 3, by Cell Signaling Technology (3 mentions)
P egfr, by Cell Signaling Technology (2 mentions)
Total akt, by Cell Signaling Technology (2 mentions)
P akt, by Cell Signaling Technology (2 mentions)
P erk1 2, by Cell Signaling Technology (2 mentions)
Bcl 2, by Agilent Technologies (2 mentions)
Coxiv, by Thermo Fisher Scientific (2 mentions)
Total erk1 2, by Cell Signaling Technology (2 mentions)
Mcl 1, by BD (2 mentions)
Bcl xl, by BD (2 mentions)
Bcl2 associated x (bax), by BD (2 mentions)
Cleaved caspase 9, by Cell Signaling Technology (2 mentions)
β actin, by Merck Group (2 mentions)
Total foxo3a, by Merck Group (2 mentions)
Total egfr, by BD (2 mentions)
Total aurora a, by Cell Signaling Technology (2 mentions)
Total aurora b, by Cell Signaling Technology (2 mentions)
P foxo3a, by Cell Signaling Technology (2 mentions)
Cytochrome c, by Santa Cruz Biotechnology (2 mentions)
Survivin, by Cell Signaling Technology (2 mentions)
8 protocols using p aurora a b c
1
Western Blot Analysis of Aurora Kinases
2
Antibodies for Cellular Signaling Analysis
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The following antibodies were used: Fyn (#25; BD Biosciences, and Fyn3; Santa Cruz Biotechnology), c-Src (#327; Calbiochem), Src[pY416] (phospho-Src family, Cell Signaling Technology), γ-tubulin (GeneTex, and clone GTU-88; Sigma-Aldrich or Abcam), phosphotyrosine (pTyr) (4G10; Upstate Biotechnology, Inc.; a gift from T. Tamura and T. Yoshimoto), IAK1 (Aurora A) (#4; BD Biosciences), phospho-Aurora A(Thr288)/Aurora B(Thr232)/Aurora C(Thr198) (pAurora A/B/C) (D13A11; Cell Signaling Technology), HA (F7 and Y-11; Santa Cruz Biotechnology), myc (9E10; Santa Cruz Biotechnology and A-14-G; Santa Cruz Biotechnology), α-tubulin (MCA78G; Serotec), cyclin B1 (H-433; Santa Cruz Biotechnology), PRC1 (H-70; Santa Cruz Biotechnology), GM130 (#35; BD Transduction Laboratories), cPLA2 (4-4B-3C; Santa Cruz Biotechnology), LAMP-1 (H4A3; Santa Cruz Biotechnology), and PDI (P7496; Sigma). Horseradish peroxidase-linked IgG F(ab’)2 and Alexa Fluor 488-, 546-, or 647-labeled IgG secondary antibodies were obtained from Amersham Biosciences and Invitrogen.
3
Comprehensive Western Blotting Analysis of Cell Signaling
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Western blotting was performed as previously described [41 (link)] using antibodies against: β-Actin (A5441, Sigma), cleaved caspase-3 (#9661, Cell Signaling, Danvers, MA, USA), cleaved caspase-9 (#9502, Cell Signaling), Mcl-1 (#559027, BD Biosciences, San Jose, CA, USA), Bax (#610983, BD Biosciences), Bid (#2002, Cell Signaling), Bcl-xL (#610212, BD Biosciences), Bim (#2819, Cell Signaling), Bcl-2 (#M0887, Agilent DAKO, Santa Clara, CA, USA), cytochrome c (sc-7159, Santa Cruz Biotechnology, Santa Cruz, CA, USA), COX IV (A21348, Invitrogen), PUMA [44 (link)], Bak (#06–536, EMD Millipore), Noxa (#OP180, EMD Millipore), p73 (A300–126A, Bethyl Laboratories, Montgomery, TX, USA), p-p73 (#4665, Cell Signaling), p-AKT (#4058, Cell Signaling), total AKT (#9272, Cell Signaling), p-ERK1/2 (#4376, Cell Signaling), total ERK1/2 (#9102, Cell Signaling), p-FoxO3A (#9464, Cell Signaling), total FoxO3A (07–702, EMD Millipore), p53 (sc-126, Santa Cruz), p-EGFR (#2234, Cell Signaling), total EGFR (#610016, BD Biosciences), KRAS (sc-30, Santa Cruz), p-Aurora A/B/C (#2914, Cell Signaling), total Aurora A (#4718, Cell Signaling), total Aurora B (#3094, Cell Signaling), and HA (#12CA5, Roche, Indianapolis, IN, USA).
4
Immunofluorescence Microscopy of DNA Damage
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Cells were placed on the slides treated with the indicated concentrations for 72 h and followed fixation with 4% paraformaldehyde in PBS for 20 min at room temperature, permeabilized with 5% Triton X-100 in PBS for 30 min. Blocked cells with 3% bovine serum albumin (BSA) were incubated with primary antibodies γ-H2AX (Cell Signaling, #9718), p-Aurora A/B/C (Cell Signaling, # 2914), Survivin (Cell Signaling, #2808), Aurora B (Santa Cruz, sc-393357), or ACA (ImmunoVision, HCT-0100) in a humidity chamber overnight. The slides were then incubated with Alexa Fluor 488 fluorochrome-conjugated secondary antibodies (Abcam, ab150077) or Alexa Fluor 647 fluorochrome-conjugated secondary antibodies (Invitrogen, A-21445) for 1h at room temperature. Stained cells were mounted with DAPI (Beyotime, China). The Nikon microscope was used to image the fluorescently-stained slides and analysis. We acquired a range of 10–20 fields per treatment using oil 100x objective magnification and at least 120 cells were analyzed using Image J software for data analysis.
5
Generation and Validation of Phospho-Specific Antibodies
6
Western Blot Analysis of Cancer Cell Response
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Cancer cells were treated with
each of the compounds at different compound concentrations. After
24 h, cells were collected, washed with 1× phosphate buffered
saline (PBS), lysed in 1× Laemmli protein sample buffer, and
boiled at 100 °C for 10 min. Each lysate was separated by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred
to polyvinylidene fluoride (PVDF, Millipore, USA) membrane, and blotted
with antibodies. Primary antibodies used for western blotting were
cMYC (Cell Signaling, 5605S), MYCN (Cell Signaling, 9405S), PARP-1(Abcam,
ab32378), GAPDH (Genetex, GTX100118), pAurora A/B/C (Cell Signaling,
#2914), Aurora A (Abcam, ab52973), pHistone H3 Ser10 (Epitomics, 1173–1),
Histone H3 (Millipore, 07-690), Cyclin B1 (Santa Cruz, sc-245), and
β-ACTIN (Sigma-Aldrich, A1978). After the blotting (0.2% casein,
1% BSA, or 5% nonfat milk), membranes were washed with blotting buffer
(0.2% casein in 1× PBS or 1× TBST), and corresponding alkaline
phosphatase (AP)- or horseradish peroxidase (HRP)-conjugated secondary
antibodies (Sigma-Aldrich) were added. The blots were developed by
chemiluminescence (PerkinElmer, USA).
each of the compounds at different compound concentrations. After
24 h, cells were collected, washed with 1× phosphate buffered
saline (PBS), lysed in 1× Laemmli protein sample buffer, and
boiled at 100 °C for 10 min. Each lysate was separated by sodium
dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred
to polyvinylidene fluoride (PVDF, Millipore, USA) membrane, and blotted
with antibodies. Primary antibodies used for western blotting were
cMYC (Cell Signaling, 5605S), MYCN (Cell Signaling, 9405S), PARP-1(Abcam,
ab32378), GAPDH (Genetex, GTX100118), pAurora A/B/C (Cell Signaling,
#2914), Aurora A (Abcam, ab52973), pHistone H3 Ser10 (Epitomics, 1173–1),
Histone H3 (Millipore, 07-690), Cyclin B1 (Santa Cruz, sc-245), and
β-ACTIN (Sigma-Aldrich, A1978). After the blotting (0.2% casein,
1% BSA, or 5% nonfat milk), membranes were washed with blotting buffer
(0.2% casein in 1× PBS or 1× TBST), and corresponding alkaline
phosphatase (AP)- or horseradish peroxidase (HRP)-conjugated secondary
antibodies (Sigma-Aldrich) were added. The blots were developed by
chemiluminescence (PerkinElmer, USA).
7
Comprehensive Western Blotting Analysis of Cell Signaling
8
Alisertib Modulates Aurora A and Jak/STAT Signaling
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The effect of alisertib on Aurora A and the Jak/STATs signaling pathways was assessed by immunoblotting, as described previously 21 (link), 23 , using the following phospho-specific antibodies: p-Aurora A/B/C (Cell Signaling Technology, #2914), Aurora A (35C1) (Abcam, ab13824), p-Jak1 (T1022/1023) (Cell Signaling Technology, #3331), Jak1 (6G4) (Cell Signaling Technology, #3344), p-Jak2 (T1007/1008) (Cell Signaling Technology, #3776), Jak2 (D2E12) (Cell Signaling Technology, #3230), p-STAT1 (T701) (Cell Signaling Technology, #7649), STAT1 (D1K9Y) (Cell Signaling Technology, #14994), p-STAT2 (T690) (Cell Signaling Technology, #4441), STAT2 (D9J7L) (Cell Signaling Technology, #72604), PARP (46D11) (Cell Signaling Technology, #9532), DNMT3B (D7070) (Cell Signaling Technology, #67259), DNMT1 (Proteintech., 24206-1-AP), DNMT3A (Proteintech., 19366-1-AP), ubiquitin (Proteintech., 10201-1-AP), UHRF1(Proteintech., 21402-1-AP), flag (Abmart, M20008L) and β-actin (Proteintech.,66009-1-Ig).
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