Anti e2f1

Manufactured by Cell Signaling Technology

Sourced in United States, Canada

Anti-E2F1 is a primary antibody that recognizes the E2F1 transcription factor. E2F1 is a key regulator of cell cycle progression and plays a role in the control of cell proliferation and apoptosis.

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39 protocols using anti e2f1

Western Blot Analysis of Signaling Proteins

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The primary antibodies used in this study included anti-Akt, anti-p-Akt, anti-CCND1, anti-E2F1, anti-p-MDM2, and anti-P27 (Cell Signaling Technology, USA) and anti-TRIB3, anti-E-cadherin, anti-MMP9, anti-H3 and anti-GAPDH (Abcam, USA), and anti-MDM2 (Proteintech, USA). The goat anti-rabbit or anti-mouse secondary antibodies were purchased from Kangwei Ltd. Beijing, China.
To determine the protein levels in cells, total protein was extracted using RIPA with a Protease Inhibitor Cocktail. Then, 30 µg of protein was subjected to 10% SDS–PAGE gel electrophoresis and transferred to a polyvinylidene fluoride membrane. After the membrane was blocked with 5% skim milk for 1 h, blots were incubated with primary antibodies. Finally, the blots were incubated with secondary antibodies after being washed with TBST.

Su F., He W., Chen C., Liu M., Liu H., Xue F., Bi J., Xu D., Zhao Y., Huang J., Lin T, & Jiang C. (2018). The long non-coding RNA FOXD2-AS1 promotes bladder cancer progression and recurrence through a positive feedback loop with Akt and E2F1. Cell Death & Disease, 9(2), 233.

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Western Blot Analysis of Protein Expression

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Anti-FLAG and anti-beta actin (Sigma-Aldrich, USA); anti-GNL1, anti-RPS20, anti-CDK1 and anti-MDM2 (Abcam, UK); anti-GFP, anti-Cyclin B1 and anti-p53 (Santa Cruz, USA); anti-p21, anti-Cyclin D1, anti-CDK4, anti-pRbSer-780, anti-Rb and anti-E2F1 (Cell Signaling Technology Inc., USA) antibodies were used in western blot analysis for checking protein expression.

Krishnan R., Boddapati N, & Mahalingam S. (2018). Interplay between human nucleolar GNL1 and RPS20 is critical to modulate cell proliferation. Scientific Reports, 8, 11421.

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Western Blot Protein Detection

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Total proteins were extracted using 1X cell lysing buffer (Cell Signaling Technology). Samples were separated by electrophoresis in SDS- polyacrylamide gels (12%) and transferred to PVDF membranes. Membranes were blocked for 1 h with PBST (PBS plus 0.1% Tween-20) containing 5% non-fat milk. Blots were incubated overnight at 4°C with primary antibodies in PBST containing 5% BSA at the manufacturer's recommended dilution. The antibodies were purchased from the following suppliers: FKBP4 (ProteinTech); anti-pS473 Akt, anti-pT308 Akt, anti-Akt, anti-pT286 Cyclin D1, anti-Cyclin D1, anti-E2F-1, anti-pT37/46 4E-BP1, anti-pS9 GSK-3β, anti-GSK-3β, and anti-mTOR (Cell Signaling Technology); anti-GAPDH and anti-Actin (Santa Cruz Biotechnology); anti-Tubulin and anti-PIK3R2 (Sigma-Aldrich). After washing, blots were incubated with either an anti-rabbit HRP, or an anti-mouse HRP-conjugated antibody (Cell Signaling Technology) for 1 h at 25°C. After washing, blots were developed with Supersignal WEST Pico Plus (Life Technologies SAS).

Mangé A., Coyaud E., Desmetz C., Laurent E., Béganton B., Coopman P., Raught B, & Solassol J. (2019). FKBP4 connects mTORC2 and PI3K to activate the PDK1/Akt-dependent cell proliferation signaling in breast cancer. Theranostics, 9(23), 7003-7015.

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E2F1 Chromatin Immunoprecipitation Assay

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M26c cells were fixed with 1% formaldehyde and lysed. DNA was sonicated and diluted with chromatin immunoprecipitation dilution buffer, and input material (5%) was collected. Chromatin was incubated overnight at 4 °C with Dynabeads Protein G (Life Technologies) pre-conjugated with anti-E2F1 (Cell Signaling) or a non-specific IgG control. DNA was purified, and qPCRs was carried out at 60 °C using Power SYBR Green PCR Master Mix (Life Technologies). Chromatin immunoprecipitation primers are reported in Supplementary Table 2.

Riverso M., Montagnani V, & Stecca B. (2017). KLF4 is regulated by RAS/RAF/MEK/ERK signaling through E2F1 and promotes melanoma cell growth. Oncogene, 36(23), 3322-3333.

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Immunoprecipitation and Western Blotting

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For immunoprecipitation (IP), cells were lysed in Triton lysis buffer supplemented with protein inhibitor cocktails (Thermo Fisher Scientific), followed by a brief sonication, and then lysates were immunoprecipitated with anti-E2F1 (Abcam), anti-LSD1 (Abcam), or anti-CoREST (Abcam) antibodies. For immunoblotting, proteins were separated on 4–15% SDS gradient gels (Bio-Rad), transferred to nitrocellulose membranes (Bio-Rad), and then probed with primary antibodies, including anti-Rb (Cell Signaling), anti-tubulin (Abcam), anti-LSD1 (Abcam), anti-CoREST (Abcam), anti-E2F1 (Cell Signaling), anti-GAPDH (Abcam), anti-V5 (Abcam), anti-H3 (Abcam) and anti-FOXA1 (Abcam).

Han W., Liu M., Han D., Li M., Toure A.A., Wang Z., Besschetnova A., Patalano S., Macoska J.A., Gao S., Hansen He H, & Cai C. (2022). RB1 loss in castration-resistant prostate cancer confers vulnerability to LSD1 inhibition. Oncogene, 41(6), 852-864.

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Inducible ERG Expression in LNCaP Cells

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LNCaP cell line was transduced with an inducible lentiviral ERG construct (LNCaP-lentivirus TMPRESS2:ERG3 inducible) to establish stable doxycycline-inducible ERG expressing LnTE3 cell line [2 (link), 16 (link)]. The cell lines were cultured in RPMI 1640, supplemented with 10% Tet System Approved Fetal Bovine Serum (Clontech Laboratories, Inc. Mountain View, CA, USA) and puromycin (Sigma, St. Louis, MO, USA) with or without doxycycline (Dox, 1 μg/ml) as per requirements and characterized as described [2 (link), 16 (link)]. Antibodies used were as follows: anti-GAPDH (Millipore MAB374), anti-ERG (Abcam ab92513), anti- p21Waf1/Cip1, anti-E2F1 and anti- c-Myc Antibody (Cell Signaling 2946, 3742 and 9402, respectively), anti-p53 DO1 (Santa Cruz biotech, sc126), and anti-NKX3.1 (Biocare Medical SKU 422).

Kumar P., Chakraborty J., Sukumar G., Dalgard C., Chatterjee R, & Biswas R. (2019). Comparative RNA-seq analysis reveals dys-regulation of major canonical pathways in ERG-inducible LNCaP cell progression model of prostate cancer. Oncotarget, 10(42), 4290-4306.

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Western Blot Analysis of Cell Signaling Proteins

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Western blot analysis was performed as described previously^{36 (link)}. The primary antibodies were as follows: anti-p27 (ab193379, Abcam), anti-phosphorylated Rb (Ser795) (#9301, Cell Signaling Technology), anti-E2F1 (#3742, Cell Signaling Technology), anti-p21 (#2947, Cell Signaling Technology), anti-caspase-3 antibody (ab32351, Epitomics), anti-FHL2 (ab12327, Abcam), anti-β-catenin (#8480, Cell Signaling Technology), anti-phosphorylated β-catenin (Ser33/37/Thr41) (#9561, Cell Signaling Technology), anti-cyclin D1 (60186-lg, Proteintech), anti-p53 (10442-1-AP, Proteintech), anti-PUMA (ab33906, Epitomics), anti-E-cadherin (20874-1-AP, Proteintech), anti-N-cadherin (22018-1-AP, Proteintech), and anti-GAPDH (#2118, Cell Signaling Technology).

Huang Z., Li Q., Luo K., Zhang Q., Geng J., Zhou X., Xu Y., Qian M., Zhang J.A., Ji L, & Wu J. (2019). miR-340-FHL2 axis inhibits cell growth and metastasis in ovarian cancer. Cell Death & Disease, 10(5), 372.

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Immunoprecipitation and Western Blotting Protocol

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Whole cell lysates were obtained by re-suspending cell pellets in RIPA buffer (50 mM Tris pH7.4, 150 mM NaCl, 1% Triton X-100) with freshly added protease inhibitor (Roche) as previously described [37 ]. Nuclear proteins were prepared with the NE-PER Kit (Pierce) following manufacturer's recommendation. Specific antibodies or pre-immune IgGs (P.I.I.) were added to and incubated with cell lysates overnight before being absorbed by Protein A/G-plus Agarose beads (Santa Cruz). Precipitated immune complex was released by boiling with 1X SDS electrophoresis sample buffer. Alternatively, FLAG-conjugated beads (M2, Sigma) were added to and incubated with lysates overnight. Precipitated immune complex was eluted with 3X FLAG peptide (Sigma). Western blotting was performed with anti-FLAG (Sigma, F3165), anti-GFP (Proteintech, 50430-1), anti-MKL1 (Proteintech, 21166-1), anti-E2F1 (Cell Signaling Tech, 3742), anti-FOXM1 (Abcam, ab207298), anti-phosphorylated serine/threonine (Cell Signaling Tech, 9631), anti-MK2 (Cell Signaling Tech, 3042), anti-phosphorylated MK2 (Cell Signaling Tech, 3316), and anti-β-actin (Sigma, A2228).

Wu T., Li N., Zhang Q., Liu R., Zhao H., Fan Z., Zhuo L., Yang Y, & Xu Y. (2022). MKL1 fuels ROS-induced proliferation of vascular smooth muscle cells by modulating FOXM1 transcription. Redox Biology, 59, 102586.

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Western Blot Analysis of Protein Markers

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A total of 25 μg protein from each sample was separated on 10% Bis-Tris polyacrylamide gel through electrophoresis and then blotted onto PVDF (polyvinylidene fluoride) membranes (Millipore). Blots were immunostained with primary antibodies, including rabbit anti-EZH2 (Cell Signaling Technology), anti-VOPP1 (Santa Cruz), anti-Ki-67 (Santa Cruz), anti-p65-NF-κB (Cell Signaling Technology), anti-E2F-1 (Cell Signaling Technology), anti-pAKT (Phospho-Thr308; Cell Signaling Technology), anti-pERK (Phospho-Thr980; Cell Signaling Technology), anti-TS (Abcam), and anti-β-actin (Cell Signaling Technology) at 4°C overnight and with secondary antibody at room temperature for 1 hr. Immunoblots were visualized using Immobilon Western Chemiluminescent HRP Substrate (Millipore). β-actin was used as the internal control.

Li P., Zhang X., Wang L., Du L., Yang Y., Liu T., Li C, & Wang C. (2017). lncRNA HOTAIR Contributes to 5FU Resistance through Suppressing miR-218 and Activating NF-κB/TS Signaling in Colorectal Cancer. Molecular Therapy. Nucleic Acids, 8, 356-369.

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Quantitative Western Blot Analysis

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Cells protein lysates were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to 0.22μm NC membranes (Sigma) and incubated with specific antibodies. Autoradiograms were quantified by densitometry (Quantity One software; Bio-Rad). β-actin antibody was used as control. Anti-E2F1 and anti-p57 were purchased from Cell Signaling Technology, Inc.

Zhang E., Yin D., Han L., He X., Si X., Chen W., Xia R., Xu T., Gu D., De W., Guo R., Xu Z, & Chen J. (2015). E2F1-induced upregulation of long noncoding RNA LINC00668 predicts a poor prognosis of gastric cancer and promotes cell proliferation through epigenetically silencing of CKIs. Oncotarget, 7(17), 23212-23226.

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