For western blot analysis, cells were harvested and extracted in lysis buffer (50 mM Tris (pH 7.4), 150 mM NaCl, 0.1% SDS, 1% NP-40, and 1 mM phenylmethylsulfonyl fluoride (PMSF)). The analysis was performed as described previously [17 ]. The primary antibodies used to detect apoptotic markers were Bcl-2 (Santa Cruz, California, CA, USA), Bax (Abcam, Cambridge, MA, USA), cytochrome C (Santa Cruz, California, CA, USA), PARP, cleaved PARP, caspase 3, cleaved caspase 3, caspase 7, cleaved caspase 7, caspase 9, and cleaved caspase 9 (all from CST, Danvers, MA, USA). The primary antibodies c-Myc, cyclin D1, and cyclin E1 (all from Abcam, Cambridge, MA, USA) were used to detect cell cycle proteins. β-Catenin, GSK3β, p-GSK3β, c-Myc, cyclin D1, and cyclin E1 (all from Abcam, Cambridge, MA, USA) were used to analyze the Wnt/β-catenin pathway. β-Actin (CST, Danvers, MA, USA) was used as an internal control.
Cyclin e1
Manufactured by Abcam
Sourced in United States, United Kingdom, China
Cyclin E1 is a protein involved in the regulation of the cell cycle. It binds to and activates cyclin-dependent kinase 2 (CDK2), which is essential for the transition from the G1 phase to the S phase of the cell cycle.
Automatically generated - may contain errors
Lab products found in correlation
Cyclin d1, by Abcam (15 mentions)
Gapdh, by Abcam (7 mentions)
β actin, by Abcam (6 mentions)
Bcl 2, by Abcam (5 mentions)
Cyclin b1, by Abcam (5 mentions)
Ripa lysis buffer, by Beyotime (4 mentions)
Bcl2 associated x (bax), by Abcam (4 mentions)
Cyclin d1, by Santa Cruz Biotechnology (4 mentions)
Cleaved caspase 3, by Cell Signaling Technology (4 mentions)
N cadherin, by Abcam (3 mentions)
Stat3, by Abcam (3 mentions)
P stat3, by Abcam (3 mentions)
Gapdh, by Proteintech (3 mentions)
Proliferating cell nuclear antigen (pcna), by Abcam (3 mentions)
β actin, by Merck Group (3 mentions)
Pvdf membrane, by Bio-Rad (3 mentions)
Cleaved caspase 3, by Abcam (3 mentions)
C myc, by Abcam (2 mentions)
Phospho rb, by Abcam (2 mentions)
β actin, by Proteintech (2 mentions)
35 protocols using cyclin e1
1
Western Blot Analysis of Apoptosis and Cell Cycle Markers
2
Protein Extraction and Western Blot Analysis
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Lysis buffer (RIPA, KeyGEN) containing protease inhibitors (PMSF, KeyGEN) was used to extract protein of cells and tissues, and protein concentration was detected with a BCA Kit (KeyGEN). Protein samples (40 μg) were loaded into 10% sodium dodecyl sulfate polyacrylamide electrophoresis (SDSPAGE) gels and transferred onto a PVDF membrane after electrophoresis. The membrane was blocked with non-fat milk for 2 h, and incubated overnight with antibodies against respective antibodies: MNX1 (Abcam, ab79541, 1:1,000); p21cip1 (Cell Signaling Technology; 2947, 1:1,000); pThr161-CDK1 (Cell Signaling Technology, 9114, 1:1,000); CDK1 (Cell Signaling Technology, 9116, 1:1,000); p27kip1 (Cell Signaling Technology, 3686; 1:1,000); CyclinB1 (Abcam, ab72, 1:1,000); CyclinE1 (Abcam, ab3927, 1:1,000); CyclinE1 (Abcam, ab3927, 1:1,000); CyclinD1(Santa Cruz Biotechnology, sc-246, 1:1,000); β-actin (Abcam, ab15265, 1:1,000).
3
Western Blot Analysis of Key Proteins
4
Cell Lysis and Western Blot Analysis
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After cells were lysed with ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology, Changzhou, China) for 30 min on ice, lysates were centrifuged at 13,000 ×g with 20 min and the supernatants were used as total cell lysates. Protein concentration was determined by Bradford protein assay (Bio-Rad, USA). A quantity of 50 μg total protein per lane was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Amersham Hybond-P polyvinylidene fluoride (PVDF) membranes (GE Healthcare, Buckinghamshire, UK). Membranes were blocked with 5% milk powder in 0.05% Tween-TBS, incubated with the specific antibodies, including Cyclin E1, p27, and CDK7 and CDC7 rabbit monoclonal antibodies (Epitomics, Burlingame, CA, USA). Detection of the target proteins on the membranes was performed using the ECL western blotting detection reagents. Signal density was detected using the MF-ChemiBIS Family including gel capture software (DNR Bio-Image system, Ltd., Jerusalem, Israel) and quantitatively analyzed by densitometry using the Image J software.
5
Protein Analysis and Epithelial-Mesenchymal Transition
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Protein preparation and Western blot analysis were performed as described previously (33 (link)). The antibodies used in the study were as follows: Chemerin (Abcam, dilution of 1:500), GAPDH, E-cadherin, N-cadherin, Vimentin, p-JAK2, JAK2, Phospho-Rb (Abcam, dilution of 1:1000), p-STAT3, STAT3, Phospho-Rb (Abcam, dilution of 1:2000), E2F1, Cyclin D1, Cyclin E1 (Abcam, dilution of 1:1000), Slug and Snail (ProteinTech, dilution of 1:1000), and β-actin (ProteinTech, dilution of 1:4000).
6
Quantitative Western Blot Analysis
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Total proteins of the cells were extracted using protein extraction kit (Beyotime). Concentrations of proteins were determined using BCA kit (Beyotime). Then, the proteins were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), followed by transferred onto PVDF membranes (Thermo Fisher). Primary antibodies against SRSF1 (1:2000, Abcam, Cambridge, MA, USA), CDK2 (1:1000, Abcam), CyclinE1 (1:1000, Abcam), β-actin (1:1000, Abcam) were incubated with the membranes at 4°C for 12 h. After washing with TBST for three times, the membranes were then incubated with corresponding HRP-conjugated secondary antibodies (1:5000, Abcam) at room temperature for 2 h. The protein samples were visualized using an ECL detection kit (Merck Millipore, Billerica, MA, USA). The integrated density of each band was normalized to the corresponding β-actin band.
7
Comprehensive Protein Expression Analysis
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Western blotting was performed as described previously [56 (link)]. The following antibodies were used for western blotting: STAT 3 (Abcam, ab109085), p-STAT 3 (phospho S727, Abcam, ab30647), p21 (Proteintech, 10355-1-AP), p27 (Proteintech, 25614-1-AP), Cyclin D1 (Abcam, ab134175), Cyclin E1 (Abcam, ab133266), Cyclin B1 (Abcam, ab181593), Cyclin A2 (Abcam, ab181591), LC3 (Cell Signalling Technology, 2775S), p62 (Abcam, ab91526), p-AMPK (Thr172, Cell Signalling Technology, 2535S), AMPK (Cell Signalling Technology, 5832T), p-mTOR (Ser2448, Cell Signalling Technology, 5536P), mTOR (Cell Signalling Technology, 2983P), BECN1 (Santa Cruz, sc-48341), ATG5 (Proteintech, 10181-2-AP), GAPDH (Proteintech, 60004-1-Ig), Cathepsin D (Cell Signalling Technology, 2284), Cathepsin B (Cell Signalling Technology, 31718), Ub (Santa Cruz, sc-8017), Caspase 8 (Proteintech, 13423-1-AP), Bid (Cell Signalling Technology, 2002T), VDAC1/2 (Proteintech, 10866-1-AP), ki67 (Abcam, ab15580), LAMP1 (Cell Signalling Technology, 9091S).
8
Diallyl Trisulfide Anticancer Mechanism
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Diallyl trisulfide (DATS, 98 %) was obtained from AIKE Regent (Chengdu, China). Sulforhodamine B (SRB), 4',6-diamidino-2-phenylindole (DAPI) and propidium iodide (PI) were purchased from Sigma-Aldrich (St. Louis, Missouri, USA). Primary antibodies against p21, p53, Bax, Bcl-2, Bcl-xl, Cytochrome C, CyclinA2, CyclinB1, CyclinD1, CyclinE1, MMP-9, PCNA and FADD were obtained from Abcam (Cambridge, United Kingdom). The antibodies against caspase-3/7/8/9, cleaved caspase-3/7/8/9, cleaved PARP, ERK, p-ERK, JNK, p-JNK, p38, p-p38 were from Cell Signaling Technology (Danver, MA, USA). Nrf2, AKT, p-AKT antibodies were from Santa Cruz Technology, Inc. (Heidelberg, Germany) and anti-E-cadherin antibody was from Millipore (Bedford, MA, USA). Other regents were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).
9
Western Blot Analysis of Protein Expression
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Cells were harvested and lysed in RIPA buffer (Sigma Aldrich) and protein concentrations were measured by a BCA protein detection kit (Pierce, Rockford, IL, USA). Proteins were separated by SDS-PAGE and transferred to nitrocellulose membranes. Primary antibodies against KLF4 (1:1000, Santa Cruz), β-actin (1:5000, Sigma Aldrich), p21 (1:1000, Cell Signaling), HA-Tag (1:1000, Santa Cruz), Cyclin E1(1:1000, Abcam), p53 (1:1000, DAKO), p16 (1:1000, Santa Cruz), Suv39H1 (1:1000, Santa Cruz), survivin (1:1000, Abcam) and horseradish peroxidase conjugated secondary antibodies (Zhongshan, Beijing, China) were used to detect specific proteins according to the standard procedures. Finally, the membranes developed with a Luminol Detection System (Santa Cruz). Protein expression was quantified using a Gel EDAS 290 analysis system (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA) and Gel-Pro Analyzer 3.1 software (Media Cybernetics, Silver Spring, MD, USA).
10
Western Blot Analysis of Cell Cycle Regulators
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Protein was extracted from the liver tissue using RIPA Lysis Buffer (Beyotime Institute of Biotechnology, Haimen, China). After centrifugation (15,000 × g, 4°C, 20 minutes), the supernatants of the homogenate were collected for protein concentration measurement. After separation by SDS-PAGE gels, the denatured protein was transferred to nitrocellulose membranes. The membranes were then incubated with primary antibodies to cyclin D1 (1:10000; Abcam), cyclin E1 (1:500; Abcam), and glyceraldehyde 3-phosphate dehydrogenase (1:1000; Abcam). The membranes were then incubated with secondary antibodies followed by use of an enhanced chemiluminescence kit (Pierce Biotechnology, Rockford, IL, USA).
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