Cyclin e1

Manufactured by Cell Signaling Technology

Sourced in United States, China

Cyclin E1 is a protein that plays a crucial role in the regulation of the cell cycle. It is a member of the cyclin family and is involved in the transition from the G1 phase to the S phase of the cell cycle.

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Rabbit anti-cyclin E1 antibody Cyclin E1 (20808) Anti-cyclin E1

114 protocols using cyclin e1

Comprehensive Antibody Panel for Cell Cycle Analysis

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The following antibodies were used in this study: myc (9E10, SC-131, Mouse monoclonal) 1:1000 WB; cyclin B1 (sc-245, Mouse monoclonal) 1:1000 WB; were from Santa Cruz Biotechnology; FADD (2782, rabbit polyclonal) 1:1000; GST (26H1, mouse monoclonal) 1:1000 WB; cyclin A2 (4656, mouse monoclonal) 1:1000 WB; cyclin E1 (4129, mouse monoclonal) 1:1000 WB; cyclin E1 (13445, rabbit monoclonal) 1:1000 WB; Phospho-Histone H3 (3377, rabbit monoclonal) 1:1000 WB; Aurora A (14475, rabbit monoclonal) 1:1000 WB; α-Actinin (6487, rabbit monoclonal) 1:1000 WB; Rb (9309, mouse monoclonal) 1:100 IF; Phospho-Rb (8516, rabbit monoclonal) 1:1000 WB, 1:350 IF; Phospho-FADD (Ser194) Antibody (Human Specific) (2781 1:1000 WB, 1:100 IF) were from Cell Signaling Technology; Cdh1 (FZR1) (MABT1323 mouse monoclonal) was from Millipore Sigma; Alexa Fluor 405 and Alexa Fluor 647 -conjugated anti-mouse-IgG and anti-rabbit-IgG secondary antibodies (A48258, A32787) were from Thermo Fisher Scientific; (HRP)-conjugated anti-mouse-IgG and anti-rabbit-IgG secondary antibodies were from Jackson Immunoresearch: horseradish peroxidase secondary antibodies (715-035-151, 711-035-152).

Awadia S., Sitto M., Ram S., Ji W., Liu Y., Damani R., Ray D., Lawrence T.S., Galban C.J., Cappell S.D, & Rehemtulla A. (2023). The adapter protein FADD provides an alternate pathway for entry into the cell cycle by regulating APC/C-Cdh1 E3 ubiquitin ligase activity. The Journal of Biological Chemistry, 299(6), 104786.

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Western Blot and Chromatin Immunoprecipitation

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For western blot analyses, cells were lysed in buffer containing 10 mM NaH₂PO₄ pH 7.2; 1 mM ethylenediaminetetraacetic acid; 1 mM Ethylene glycol tetraacetic acid (EGTA); 150 mM NaCl; 1% NP-40, and a cocktail of protease and phosphatase inhibitors (Roche). Protein concentrations in supernatants were determined using a commercially available kit (DC Protein Assay from Bio-Rad). A total of 20 μg of protein were loaded per lane, fractionated in 8–10% sodium dodecyl sulphate-polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad). Antibodies against the following proteins were used: E2F7 (sc-32574, Santa Cruz), Cyclin E1 (4129, Cell Signaling), p53 (sc-1312, Santa Cruz), RAD51 (sc-8349, Santa Cruz), pH3 (06-570, Millipore), α-Tubulin (T-9026, Sigma), β-Actin (A5441, Sigma). Immunocomplexes were visualized with horseradish peroxidase-conjugated anti-mouse, anti-goat or anti-rabbit IgG antibodies (Santa Cruz), followed by chemiluminiscence detection (ECL, Amersham) with a ChemiDoc camera (Bio-Rad).
Chromatin immunoprecipitations (ChIPs) and the quantification of immunoprecipitated DNA sequences by qPCR were performed as described previously (25 (link)). Sequences of qPCR primers are listed in Supplementary Table S3. Antibodies used for ChIP analysis were: E2F7 (sc-66870, Santa Cruz), and SV40LT (sc-147, Santa Cruz).

Mitxelena J., Apraiz A., Vallejo-Rodríguez J., García-Santisteban I., Fullaondo A., Alvarez-Fernández M., Malumbres M, & Zubiaga A.M. (2018). An E2F7-dependent transcriptional program modulates DNA damage repair and genomic stability. Nucleic Acids Research, 46(9), 4546-4559.

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Detecting IMUP Interactome via IP-MS

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Antibodies against IMUP (1:500; #ab228823), pCDC25A (Phospho S124; 1:1000; #ab156574), pCHK1 (1:500; ab79758), from Abcam; cyclin-dependent kinase 2 (CDK2) (1:1000; #AF6237) from Affinity Biosciences (OH, USA); Cyclin A2 (1:1000; #BF683) and Cyclin E1 (1:1000; #HE12) from Cell Signaling Technology (Danvers, MA, USA); and FHL1 (1:500; #10,991–1-AP), CDC25A (1:1000; #55,031–1-AP), CHK1 (1:1000; #25,887–1-AP), CDC25C (1:1000; #16,485–1-AP), 14–3-3ξ (1:1000; #11,648–2-AP), SP1 (1:1000; #21,962–1-AP), and NPM1 (1:1000; #10,306–1-AP) from Proteintech were used for WB assays.
Antibodies against IMUP (1:100; #ab228821) from Abcam; GFP-Trap® Magnetic Agarose (20 μL per reaction; #gtma-100) from Proteintech; and Pierce™ anti-DYKDDDDK Magnetic Agarose (20 μL per reaction; #A36798) from Invitrogen were used for IP to detect protein interactions. For LC/MS, immunocomplexes from GFP-Trap® Magnetic Agarose, anti-IMUP agarose, or anti-DYKDDDDK magnetic agarose were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Subsequently, the candidate bands were cut and identified by LC/MS.

Luo Q., Pan Y., Fu Q., Zhang X., Zhou S., Yu P., Tian H., Liu P., Chen S., Zhang H, & Qin T. (2022). Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity. Cell Biology and Toxicology, 39(5), 2069-2087.

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Western Blot Analysis of Cell Cycle Proteins

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Total protein and immunoprecipitated samples were resolved by SDS-PAGE and transferred to PVDF membrane, blocked with 5% non-fat milk or BSA for 1 h at RT and incubated overnight at 4°C with primary antibody (TPC1 and TPC2, Bethyl Laboratories; cyclin B1, phospho-cdc2, phospho-Rb, cyclin E1, cyclin A, phospho-p44/42 MAPK, phospho-MLC2, Cell Signaling Technology) diluted in antibody diluent (15 mM Tris base, 150 mM NaCl, 0.05% Tween-20, 0.05% NaN₃). The membrane was washed in TTBS (15 mM Tris base, 150 mM NaCl, 0.05% Tween-20) and incubated with HRP-conjugated IgG antibodies (GE Healthcare, 1:12,000) in 0.5% non-fat milk at RT for 45 min before visualization with ECL Plus detection reagent (GE Healthcare) on a Kodak X-OMAT 2000A processor with Kodak X-OMAT LS imaging film. Densitometry analysis was performed using ImageJ software.

Horton J.S., Wakano C.T., Speck M, & Stokes A.J. (2015). Two-pore channel 1 interacts with citron kinase, regulating completion of cytokinesis. Channels, 9(1), 21-29.

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Immunoblotting of cell signaling proteins

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Proteins were extracted from WT9-7 cells using RIPA lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.5% deoxycholate, 1% Nonidet P-40, 0.1% SDS, 1 mM phenylmethylsulfonyl fluoride and protease cocktail at 1 μg/mL). The protein concentrations were determined using a BCA kit (Pierce, USA). Protein samples (50 μg per lane) were separated by 6%, 8%, 10% or 12% sodium dodecyl sufate–polyacrylamide gel electrophoresis (SDS-PAGE) electrophoresis and transferred to nitrocellulose (NC) membranes. After Ponceau S staining, the membranes were incubated overnight in 5% non-fat milk at 4 °C and then incubated with primary antibodies against p-AMPKα, p-mTOR, p-4E-BP1, p-p70 S6 kinase, p-PKA, p-PI3 kinase p85, p-Akt, p-B-Raf, p-MEK1/2, p-p44/42 MAPK (Erk1/2), PCNA, cyclin E1, Cdk2, Cdk4, (Cell Signaling Technology, USA), cyclin D3 (Wuhan Sanying Biotech Co., Ltd., China) and β-actin (Sigma, USA). Immunoreactive bands were visualized using ECL reagent (KeyGen Biotech Co., Ltd., China), according to the manufacturer’s instructions, and exposure to X-ray film. The protein band intensities were quantified using ImageJ software (NIH, Bethesda, USA).

Zhao J., Ma Y., Zhang Y., Fu B., Wu X., Li Q., Cai G., Chen X, & Bai X.Y. (2019). Low-dose 2-deoxyglucose and metformin synergically inhibit proliferation of human polycystic kidney cells by modulating glucose metabolism. Cell Death Discovery, 5, 76.

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Western Blot Protein Analysis Protocol

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The middle section of the graft was lysed in Radio Immunoprecipitation Assay (RIPA) buffer (Beyotime, Shanghai, China). BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, USA) was used to test the concentration of proteins. Total proteins (20 μg) were subjected to 6–10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels and transferred to the polyvinylidene fluoride (PVDF) membranes. The membranes were cultured in 5% bovine serum albumin (BSA; Solarbio, Beijing, China) solution for 1 h and sequentially incubated with the primary antibodies overnight at 4°C, such as Skp2 (#4358), P27 (#3686), c-Myc (#5605), CyclinE1 (#20808) and GAPDH (#8884, all from Cell Signaling Technology, Danvers, USA). The membranes were washed three times with TBST for 5 min each time, and then the HRP-labelled secondary antibodies (ab191866, 1 mg/ml, Abcam, Cambridge, USA) were added and incubated for 1 h at room temperature. Specific signals were detected using the ECL western blotting detection system.

Qiu J., Shu C., Li S., Xiong Q., Wang L., Liu Z., Li X, & Zhang W. (2021). Radiotherapy inhibits neointimal hyperplasia after artificial vascular replacement through Skp2/P27kip1. Journal of Radiation Research, 63(1), 36-43.

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Src and Akt Signaling Pathway Assay

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PP2 was purchased from MCE (MedChemExpress, Monmouth Junction, NJ, USA) and dissolved in DMSO (Sigma‐Aldrich, St Louis, MO, USA). Anti‐human Src, p‐Src (Tyr416), Akt, p‐Akt (Thr308), GAPDH, CDK4, cyclin D1, cyclin E1, vimentin, E‐cadherin, and N‐cadherin antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA). Anti‐human Snail antibody was purchased from Abcam (Cambridge, MA, USA). Anti‐mouse and anti‐rabbit antibodies were from Jackson Immunoresearch (West Grove, PA, USA).

Lou L., Yu Z., Wang Y., Wang S, & Zhao Y. (2018). c‐Src inhibitor selectively inhibits triple‐negative breast cancer overexpressed Vimentin in vitro and in vivo. Cancer Science, 109(5), 1648-1659.

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Western Blot Analysis of Cell Lysates

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For western blot analyses, cells were lysed in buffer containing 10 mM NaH₂PO₄ pH 7.2; 1 mM EDTA; 1 mM EGTA; 150 mM NaCl; 1% NP-40 and a cocktail of protease and phosphatase inhibitors (Roche). Protein concentrations in supernatants were determined using a commercially available kit (DC Protein Assay from Bio-Rad). A total of 20 μg of protein were loaded per lane, fractionated in 8–10% sodium dodecyl sulphate-polyacrylamide gels and transferred onto nitrocellulose membranes (Bio-Rad). Antibodies against the following proteins were used: E2F7 (sc-32574, Santa Cruz), Cyclin E1 (4129, Cell Signaling), c-MYC (sc-42, Santa Cruz), LIN28B (4192, Cell Signaling), HA (MMS-101R, Covance), p-H3 (06-570, Millipore), α-Tubulin (T-9026, Sigma), β-Actin (A5441, Sigma). Immunocomplexes were visualized with horseradish peroxidase-conjugated anti-mouse, anti-goat or anti-rabbit IgG antibodies (Santa Cruz), followed by chemiluminiscence detection (ECL, Amersham) with a ChemiDoc camera (Bio-Rad).

Mitxelena J., Apraiz A., Vallejo-Rodríguez J., Malumbres M, & Zubiaga A.M. (2016). E2F7 regulates transcription and maturation of multiple microRNAs to restrain cell proliferation. Nucleic Acids Research, 44(12), 5557-5570.

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Silibinin and NAC Mechanism Study

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Silibinin (purity >98%, as assessed by high-performance liquid chromatography) and N-acetyl-L-cysteine (NAC) were procured from Sigma-Aldrich (St. Louis, MO, USA). Dimethyl sulfoxide (DMSO) was used to dissolve Silibinin during in vitro experiments. Primary antibodies against Bcl-2 (#3498), cleaved PARP (#5625), Bax (#5023), cyclin D1 (#2922), cleaved caspase-3 (#9661), cyclin-dependent kinase4 (CDK4) (#12790), CDK6 (#3136), cyclin E1 (#4129), E-cadherin (#3195), N-cadherin (#4061), , p-AKT (#9271), AKT (#9272), p-ERK (#9101), ERK (#9102), p-p38 (#9216), p38 (#9212), cyclin D1 (#2922), p-c-Jun (#3270S), and c-Jun (#9165) were procured from Cell Signaling Technology (Danvers, MA, USA). And p53 (sc-1312), SOD1 (sc-11407), SOD2 (sc-18503), β-Actin (sc-47778) was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Also, vimentin (ab92547) was obtained from abcam.

Zhang H., Kim H., Kim S.Y., Hai H., Kim E., Ma L., Kim D., Kim C.Y., Park K., Park S., Ko J., Kim E.K., Kim K., Ryoo Z.Y., Yi J, & Kim M.O. (2023). Silibinin induces oral cancer cell apoptosis and reactive oxygen species generation by activating the JNK/c-Jun pathway. Journal of Cancer, 14(10), 1875-1887.

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Western Blot Analysis of Cellular Signaling

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Cells were lysed in PRO-PREP solution (Intron). Total protein concentrations were measured using a BCA protein assay kit (Thermo Scientific, cat# 23225). Samples were run onto SDS-polyacrylamide gels and transferred onto nitrocellulose membranes (Pall Corporation, Port Washington, NY). After blocking with 5% skim milk (w/v), the membranes were incubated with primary antibodies. Blots were then incubated with peroxidase-conjugated secondary antibodies and visualized using enhanced chemiluminescence (Intron). The following primary antibodies were used: PCNA (cat# SC-7907), NQO1 (cat# SC-32793), p-ERK (cat# SC-7383), and β-Actin (cat# SC-47778) (Santa Cruz Biotechnology, Santa Cruz, CA); Cyclin D1 (cat# 2978S), Cyclin E1 (cat# 4129S), SOX2 (cat# 2748S), E-cadherin (cat# 3195S), N-cadherin (cat# 4061S), Vimentin (cat# 5741S), Snail (cat# 3895S), p-JNK (cat# 4668S), p-AKT (cat# 4060), and p-p38 (cat# 9216S) (Cell Signaling Technology, Danvers, MA); and p63 (cat# ab53039) and Slug (cat# ab27568) (Abcam, Cambridge, UK).

Zhang Q.L., Li X.M., Lian D.D., Zhu M.J., Yim S.H., Lee J.H., Jiang R.H, & Kim C.D. (2019). Tumor Suppressive Function of NQO1 in Cutaneous Squamous Cell Carcinoma (SCC) Cells. BioMed Research International, 2019, 2076579.

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