Western blot analyses were performed as described previously and repeated twice.14 (link),32 (link) Primary antibodies used in this study include rabbit polyclonal antibodies to human NS (Ab138, Tsai Laboratory, Houston, TX, USA), p-cdc2 (Y15, Cell Signaling, Danvers, MA, USA), MDM2 (SMP-14, Santa Cruz, Santa Cruz, CA, USA), p53 (DO-1, Santa Cruz), p-histone H3 (S10, Upstate, Lake Placid, NY, USA), and α-tubulin (Sigma). Secondary antibodies were conjugated to peroxidase (Jackson ImmunoResearch, West Grove, PA, USA).
P cdc2 y15
Manufactured by Cell Signaling Technology
Sourced in United States
P-cdc2 (Y15) is an antibody that detects phosphorylation of Cdc2 (also known as CDK1) at tyrosine 15. Cdc2 is a key cell cycle regulatory protein that plays a critical role in the G2/M transition.
Automatically generated - may contain errors
Lab products found in correlation
α tubulin, by Merck Group (2 mentions)
Mdm2 smp 14, by Santa Cruz Biotechnology (2 mentions)
P53 do 1, by Santa Cruz Biotechnology (2 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (2 mentions)
Anti phistone h3 s10, by Merck Group (2 mentions)
Ph2ax s139, by Cell Signaling Technology (2 mentions)
Cyclin b1, by Cell Signaling Technology (2 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
P erk1 2, by Cell Signaling Technology (1 mentions)
Gapdh, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Pchk2 t68, by Cell Signaling Technology (1 mentions)
Pcdc25c s216, by Cell Signaling Technology (1 mentions)
Pakt s473, by Cell Signaling Technology (1 mentions)
Aurora b, by Cell Signaling Technology (1 mentions)
Survivin, by Cell Signaling Technology (1 mentions)
P chk1, by Cell Signaling Technology (1 mentions)
P atr, by Cell Signaling Technology (1 mentions)
P h3s10, by Cell Signaling Technology (1 mentions)
P atm, by Cell Signaling Technology (1 mentions)
8 protocols using p cdc2 y15
1
Western Blot Analysis of Cellular Proteins
2
Western Blot Analysis of Cell Signaling
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Anti-pHistone H3 (S10) was obtained from Millipore; Chk1, pChk1 (S317), pChk1 (S345), pChk2, pChk2 (T68), pCdc25c (S216), 53BP1, Cdc2, pCdc2 (Y15), Cyclin B1, D1 and E, PARP, pERK1/2, ERK 1/2, AKT, pAKT (S473), Bcl-XL, GAPDH and pH2AX (S139) from Cell Signaling Technologies; pChk1 (S296), FANCF and FANCD2 from Abcam, and Bcl-2 and Mcl-1 from Santa Cruz. Treated and untreated cells were washed once with PBS and lysed in RIPA buffer containing protease and phosphatase inhibitor cocktails (Roche). Protein concentration was determined using BCA kit (Pierce). Equal amounts of lysate were separated by SDS-PAGE and western blot analysis conducted using the antibodies indicated above.
3
Synthesis and Characterization of Anticancer Agents
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MPT0G211, tubastatin A (TBA), and SAHA were synthesized by Dr. Jing-Ping Liou’s Lab. (School of Pharmacy, College of Pharmacy, Taipei Medical University, Taiwan), and the purity are more than 98%. Doxorubicin (DOXO), cyclophosphamide (CTX), and vincristine (VCR) was obtained from Cayman Chemical (Ann Arbor, MI, USA). Antibodies against BCL-2, BCL-XL, cleaved caspase 3, caspase 8, caspase 9, acetyl-α-Tubulin, acetyl-histone 3, histone 3, HDAC6, survivin, p-ATM, p-ATR, p-CHK1, CHK1, cyclin B1, aurora B, p-PLK1, p-H3S10, p-CDC2 (Y15), and p-CDC2 (T161) were purchased from Cell signaling (Danvers, MA, USA). α-Tubulin, γ-H2AX, ATM, ATR, BAX, cytochrome c, and COX IV were from Genetex (Irvine, CA, USA). PARP and CDC2 were from Santa Cruz Biotechnology (Dallas, TX, USA).
4
Cell Cycle Regulation Protein Analysis
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Anti- pHistone H3 (S10) was obtained from Millipore; Chk1, pChk1 (S317), pChk1 (S345), Cdc2, pCdc2 (Y15), Cyclin B1 and pH2AX (S139) from Cell Signaling Technologies and pChk1 (S296) from Abcam. Treated and untreated cells were washed once with PBS and lysed in 50 mM Tris-pH6.8, 2% SDS, protease and phosphatase inhibitor cocktails (Roche) and boiled for 5 minutes. Protein concentration was determined using BCA kit (Pierce). Equal amounts of lysate were separated by SDS-PAGE and western blot analysis conducted using the antibodies indicated above.
5
Western Blot Analysis of Protein Expression
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Protein samples from unexpressed and stably re-expressed KYSE150 and KYSE450 cells were collected and western blots were performed as described previously [44 (link)]. Antibodies were diluted according to the manufacturer's instructions. Primary antibodies included DACT2 (Cat: TA306668, OriGene Tech, MD, USA), c-Myc (Cat: 10828-1-AP, proteintech, IL, USA), cyclin B1 (Cat: 55004-1-AP, proteintech, IL, USA), CDC2 (Cat: 19532-1-AP, proteintech, IL, USA), p-CDC2 (Y15) (Cat: #9111, Cell Signaling Tech, MA, USA), MMP2 (Cat: BS1236, Bioworld Tech, MN, USA), MMP9 (Cat: BS1241, Bioworld Tech, MN, USA), Anti-Active-β-Catenin, clone8E7 (Cat: #05-665, Millipore, CA, USA), p-β-catenin (S37) (Cat: BS4739, Bioworld Tech, MN, USA), β-catenin (D10A8) XP® (Cat: #8480, Cell Signaling Tech, MA, USA) and β-actin (Cat: AF0003, Beyotime Biotech, Jiangsu, China).
6
Chk1, Cdc2, and β-actin Detection
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Chk1, Chk1 phosphorylated at Ser345 (p-Chk1S345) or Ser296 (p-Chk1S296), Cdc2 phosphorylated at Tyr15 (p-Cdc2Y15), and β-actin were detected by Western blotting. Briefly, cells were lysed using the Mammalian Protein Extraction Reagent (M-PER) (Pierce, Rockford, USA), and equal amounts of protein from cell lysates were separated using SDS-PAGE. Proteins were transferred to nitrocellulose membranes, and the membranes were blocked in 2% ECL advance blocking agent (GE Healthcare, Uppsala, Sweden) in Tris-buffered saline with Triton X-100. Proteins were detected using specific primary antibodies against Chk1 (Sigma-Aldrich), p-Chk1S345, p-Chk1S296, p-Cdc2Y15 (Cell Signaling Technology, Danvers, MA, USA), and β-actin (clone C4; Millipore, Billerica, MA, USA). Specific proteins were visualized using secondary antibodies conjugated with horseradish peroxidase (Santa Cruz Biotechnology, Dallas, USA) and the ECL Western Blotting Detection reagents (GE Healthcare, Buckinghamshire, UK).
7
Western Blot Analyses of Key Proteins
8
Protein Expression Analysis in HeLa Cells
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HeLa cells were incubated in the presence of the test compound and, after different times, were collected, centrifuged, and washed two times with ice cold phosphate buffered saline (PBS). The pellet was then resuspended in lysis buffer. After the cells were lysed on ice for 30 min, lysates were centrifuged at 15000 x g at 4 °C for 10 min. The protein concentration in the supernatant was determined using the BCA protein assay reagents (Pierce, Italy). Equal amounts of protein (10 μg)
were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Criterion Precast, BioRad, Italy) and transferred to a PVDF Hybond-P membrane (GE Healthcare). Membranes were blocked with a bovine serum albumin solution (5% in Tween PBS 1X), the membranes being gently rotated overnight at 4 °C. Membranes were then incubated with primary antibodies against caspase-9 cleaved fragment, PARP, cdc25c, cyclin B, p-cdc2 Y15 (all from Cell Signaling), or vinculin (Sigma-Aldrich) for 2 h at room temperature. Membranes were next incubated with peroxidase labeled secondary antibodies for 1 h. All membranes were visualized using ECL Select (GE Healthcare), and images were acquired using a Uvitec-Alliance imaging system (Uvitec, Cambridge, UK). To ensure equal protein loading, each membrane was stripped and reprobed with an anti-vinculin antibody.
were resolved using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Criterion Precast, BioRad, Italy) and transferred to a PVDF Hybond-P membrane (GE Healthcare). Membranes were blocked with a bovine serum albumin solution (5% in Tween PBS 1X), the membranes being gently rotated overnight at 4 °C. Membranes were then incubated with primary antibodies against caspase-9 cleaved fragment, PARP, cdc25c, cyclin B, p-cdc2 Y15 (all from Cell Signaling), or vinculin (Sigma-Aldrich) for 2 h at room temperature. Membranes were next incubated with peroxidase labeled secondary antibodies for 1 h. All membranes were visualized using ECL Select (GE Healthcare), and images were acquired using a Uvitec-Alliance imaging system (Uvitec, Cambridge, UK). To ensure equal protein loading, each membrane was stripped and reprobed with an anti-vinculin antibody.
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