Western Blotting analysis was performed as described previously 32 (link). Total cell extracts were lysed in cell lysis buffer (Beyotime, P0013), and the lysates were centrifuged at 12,000 rpm for 10 min at 4°C. Samples containing 30 µg protein were loaded on SDS-PAGE gels and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk or BSA and probed with primary antibodies and horseradish peroxidase-conjugated secondary antibodies. Blots were developed with lumi-light Western blotting substrate detection reagents (Roche, 12015200001). Nuclear- and cytoplasmic-enriched fractions were prepared using Nuclear-Cytosol Extraction Kit (Applygene, P1200), according to manufacturer's instructions. Western Blotting analysis was performed using the anti-p27 (Cell Signaling Technology, 3698S), anti-CREB (Cell Signaling Technology, 9197L), anti-phospho-CREB (Ser133) (Cell Signaling Technology, 9198L), anti-S-phase kinase-associated protein 2 (Skp2) (Cell Signaling Technology, 4313S), anti-CDK2 (Cell Signaling Technology, 2546S), anti-Cyclin E1 (Cell Signaling Technology, 4129S), PARP (Cell Signaling Technology, 9532S), anti-GAPDH (Cell Signaling Technology, 2118L), anti-β-arrestin 1 (Abcam, ab32099), anti-β-arrestin 2 (Abcam, ab54790), anti-tubulin (Beyotime, AT819), anti-cyclin D3 (Beyotime, AC856), anti-p21 (Beyotime, AP021).
Anti cyclin e1
Manufactured by Cell Signaling Technology
Sourced in United States
Anti-cyclin E1 is a primary antibody that specifically binds to cyclin E1, a key regulator of the cell cycle. Cyclin E1 is involved in the transition from the G1 phase to the S phase of the cell cycle. The Anti-cyclin E1 antibody can be used in various applications, such as Western blotting, immunoprecipitation, and immunohistochemistry, to detect and study the expression and function of cyclin E1 in biological samples.
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Lab products found in correlation
Anti cyclin d1, by Cell Signaling Technology (13 mentions)
Anti cdk2, by Cell Signaling Technology (6 mentions)
Anti cyclin b1, by Cell Signaling Technology (5 mentions)
Anti β actin, by Cell Signaling Technology (5 mentions)
Anti gapdh, by Cell Signaling Technology (4 mentions)
Anti p21, by Cell Signaling Technology (4 mentions)
Anti cdk4, by Cell Signaling Technology (4 mentions)
Anti cdk6, by Cell Signaling Technology (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Pvdf membrane, by Merck Group (3 mentions)
Anti erk1 2, by Cell Signaling Technology (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
Anti p27, by Cell Signaling Technology (2 mentions)
Anti vimentin, by Cell Signaling Technology (2 mentions)
Anti p62, by Cell Signaling Technology (2 mentions)
Anti beclin 1, by Cell Signaling Technology (2 mentions)
Anti lc3a b, by Cell Signaling Technology (2 mentions)
Anti akt, by Cell Signaling Technology (2 mentions)
Anti phospho akt, by Cell Signaling Technology (2 mentions)
Anti cyclin d2, by Cell Signaling Technology (2 mentions)
23 protocols using anti cyclin e1
1
Comprehensive Western Blotting Analysis
2
Immunofluorescence Analysis of DNA Damage Markers
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Antibodies used in the study: anti-53BP1 (dilution: 1:500, sc-629, Santa Cruz Biotechnology), anti-γH2AX (dilution: 1:1000, 05-636, Upstate Biotechnology, Millipore), secondary antibodies (dilution: 1:600, anti-mouse Alexa 568, A11004, anti-rabbit Alexa 488, A11008, Molecular Probes), anti-pimonidazole (mouse monoclonal 4.3.11.3, Natural Pharmacia International, Belmont, MA, USA, dilution 1:100), anti-BrdU (mouse monoclonal, Clone Bu20a, Dako Deutschland GmbH, Hamburg, Germany, dilution: 1:50), anti-pRB (Ser807/811, 1/1000) (Cell signaling, #9308), anti-Cyclin E1 (Cell signaling, 20808, 1/1000), anti-HIF1a (Cayman Chemicals 10006421, 1/1000), anti-alpha-tubulin (Sigma Aldrich T5168, 1/1000). Reagents used in the study: Doxycycline (D9891, Sigma-Aldrich), nocodazole (M1404, Sigma-Aldrich), Hoechst 33342 (B2261, Sigma-Aldrich), BrdU (Sigma 850187), pimonidazole (Hypoxyprobe Inc, hpi, Middlesex, Burlington, USA), SirDNA kit (SPIROCHROME), AEC kit (Signa Aldrich AEC 101), Dako Faramount aqueous mounting medium (S3025).
3
Comprehensive Protein Expression Analysis
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Western blotting was performed according to protocol. Briefly, cells were lysed in RIPA buffer. Cellular proteins were collected and subjected to 10% SDS-PAGE and transferred onto PVDF membranes. The membranes were then incubated with specific primary antibodies (anti-cyclin B1 (#12231, Cell Signaling Technology, USA), anti-cyclin D1(#55506, Cell Signaling Technology, USA), anti-cyclin E1 (#20808, Cell Signaling Technology, USA), anti-N-cadherin (#13116, Cell Signaling Technology, USA), anti-vimentin (#5741, Cell Signaling Technology, USA), anti-TK1 (#28755, Cell Signaling Technology, USA), and anti-GAPDH (#2118, Cell Signaling Technology, USA)) overnight at 4°C. After the membranes were incubated with secondary antibodies, they were subjected to immunoblot analysis using an ECL immunoblotting kit according to the manufacturer's protocol.
4
Immunoblotting for Cyclin E1
5
Comprehensive Western Blot Analysis
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Western blot assay was carried out as previously described (Wang et al., 2019 (link)). The primary antibodies, including anti-CBS, anti-CSE, anti-3-MST, anti-cyclin E1, anti-cyclin D1, anti-cyclin-dependent kinase-2 (CDK2), anti-cyclin-dependent kinase-4 (CDK4), anti-p27, anti-p21, anti-beclin-1, anti-LC3A/B, anti-P62, anti-phosphatidylinositol 3-kinase (PI3K), anti-AKT, anti-mammalian target of rapamycin (mTOR), anti-phospho (p)-PI3K (Tyr199/Tyr458), anti-p-AKT (Ser473), and anti-p-mTOR (Ser2448) were purchased from Cell Signaling Technology (CST, Danvers, MA, United States). The primary antibodies, including anti-cleaved caspase (cas)-3, anti-cleaved cas-9, anti-cleaved poly adenosine diphosphate-ribose polymerase (PARP), anti-β-actin, and the horseradish peroxidase-conjugated secondary antibody were purchased from Proteintech (Chicago, IL, United States). The immunoreactive bands were visualized by a chemiluminescence detection system (Thermo Fisher, Waltham, MA, United States).
6
Cell Signaling Protein Analysis Protocol
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Transfection reagents Lipofectamine 2000 and Lipofectamine 3000 were purchased from Invitrogen, RNA synthesis inhibitor actinomycin D, protease inhibitor MG132, lysosomal inhibitors bafilomycin and hydroxychloroquine, CDK inhibitor RO-3306, and thymidine were purchased from Sigma-Aldrich. λPPase was purchased from New England BioLabs. Anti-CELF6 (ab173282) and anti-GFP (ab1218) were obtained from Abcam, anti-p21Waf1/Cip1 (#2947), anti-p27Kip1(#3686), anti-Gadd45α (#4632), anti-Cyclin B1(#12231), anti-β-TrCP (#4394), anti-Wee1 (#13084), anti-cleaved PARP (#5625), anti-cleaved Casp3 (#9661), anti-cyclin E1 (#20808), and anti-Ubiquitin (#3936) antibodies were purchased from Cell Signaling Technology, anti-P53 (10442-1-AP), anti-GFP (66002-1-1 g), anti-His (66005-1-1 g), anti-β-tubulin (60008-1-1 g), and anti-GAPDH (10494-1-AP) were obtained from Proteintech, anti-LC3B (L7543) was obtained from Sigma-Aldrich.
7
Mechanism of Herbal Formulation's Anti-Proliferative Effect
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Ginseng Radix, Pinellia Tuber, Bupleurum Root, Glycyrrhizae Radix et Rhizoma, Scutellaria Root, Zingiberis Rhizoma Crudus and Zizyphi Fructus were purchased from Yeongcheon herbal market (Yeongcheon, Korea). Identification of all plant material was confirmed by Prof. Ki Hwan Bae of the College of Pharmacy, Chungnam National University (Daejeon, Korea), and all voucher specimens were deposited in the herbal bank in Korea Institute of Oriental Medicine (KIOM, Daejeon, Korea). Dulbecco’s Modified Eagle Medium (DMEM) was purchased from Lonza (Wakersville, MD, USA). Fetal bovine serum (FBS) and phosphate-buffered saline (PBS) were purchased from Hyclone (Longan, UT, USA). Penicillin/streptomycin and trypsin/EDTA were purchased from Gibco (Grand Island, NY, USA). Anti-phospho-ERK1/2, anti-phospho-Akt, anti-phospho-PLCγ1, anti-ERK1/2, anti-Akt, anti-PLCγ1, anti-CDK2, anti-CDK4, anti-cyclin D1, anti-cyclin E1 and anti-β-actin antibodies were from Cell Signaling Technology Inc. (Beverly, MA). Anti- phospho-proliferating cell nuclear antigen (PCNA) was purchased from Abfrontier (Seoul, Korea). PDGF-BB was obtained from Upstate Biotechnology (Lake Placid, NY, USA). Cell Counting Kit-8 (CCK-8) was purchased from Dojindo Molecular Technologies (Rockville, MD, USA). Other chemicals were of analytical grade.
8
Comprehensive Molecular Signaling Analysis
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All the antibodies used in the present study, including anti-MEK (catalog number, 4694) and anti-ERK (catalog number, 9102) with their Phospho-antibody (catalog number, 3598 and 4370 respectively), anti-Ras (catalog number, 3965), anti-cyclinD1 (catalog number, 2922), anti-cyclinE1 (catalog number, 4129), anti-cleaved caspase-3 (catalog number, 9661) and anti-cleaved pARP1 (catalog number, 5625), anti-β-actin (catalog number, 3700), were purchased from Cell Signaling Technology. Antibody against BTG2 was purchased from Santa Cruz Biotechnologies (catalog number, sc-33775). Dual Luciferase Reporter Gene Assay Kit was purchased fromBeyotime Biotechnology. TUNEL FITC Apoptosis Detection Kit and Cell Cycle Assay Kit were purchased from Vazyme. PCR Reagents and restriction endonucleases were purchased from Takara. PRL-TK vectors and pmirGLO Vector were purchased from Promega. PEGFP-C1 was purchased fromClontech Laboratories Inc and PcDNA 3.1(+) was purchased from Invitrogen. The miRNA mimics and inhibitor were purchased from Biotend Biotechnologies.
9
Protein Expression Analysis by Western Blot
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After treatment, the liquid supernatant was taken out and the cells were washed thrice in the surface of 6-well plates. Then, cell lysates were prepared and the concentration of protein was quantified by using BCA protein assay kit (ThermoFisher, USA). Equal amount of proteins was subjected to 10% or 12% SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes (Millipore, Bedford, MA, USA). Membranes were blocked and probed with specific antibodies (dilution ratio: 1:3000, anti-cyclin A2, anti-cyclin E1, anti-cyclin D2, anti-p-cdc2, anti-CDK2, anti-CDK4, anti-Bax, anti-Bcl-xl, anti-p62, anti-Atg-5, anti-Beclin-1, anti-LC3A/B, and anti-β-actin, Cell Signaling Technology, Danvers, MA, USA) followed by exposure to a horseradish peroxidase–conjugated goat anti-mouse or goat anti-rabbit antibody and secondary antibodies (dilution ratio: 1:5000, Cell Signaling Technology, Danvers, MA, USA). The immunocomplexes were visualized using a horseradish peroxidase-conjugated antibody, followed by a chemiluminescence reagent (Millipore, Bedford, MA, USA) and detected on photographic film.
10
Immunoblotting for Stemness and Cell Cycle
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For immunoblotting, equal amounts of proteins (30∼80μg) were separated on 10%–12% SDS-PAGE and were electrophoretically transferred onto PVDF membranes (Millipore), which were blocked in TBST containing 5% defatted milk for 1 hour at RT and then blotted with antibody overnight at 4°C: anti-Nanog (1:1000, Cell signaling), anti-Oct4 (1:1000, Cell signaling), anti-Sox2 (1:1000, Cell signaling), anti-CyclinD1 (1:1000, Cell signaling), anti-CyclinE1 (1:1000, Cell signaling), anti-E2F1 (1:1000, Cell signaling), anti-CDK2 (1:1000, Cell signaling), anti-CDK6 (1:1000, Cell signaling) and anti-β-actin (1:1000, Cell signaling). After being washed with TBST and incubation with either anti-rabbit or anti-mouse horseradish peroxidase-conjugated secondary antibody (1:5000, Promoter Wuhan) in TBST containing 5% nonfat milk, immune complexes were visualized using the Beyo ECL Plus.
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