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E2f1 c20

Manufactured by Santa Cruz Biotechnology
Sourced in United States

E2F1 (C20) is a laboratory reagent product offered by Santa Cruz Biotechnology. It is an antibody that specifically targets the E2F1 protein, which is a transcription factor involved in the regulation of cell cycle progression and proliferation. The product is intended for use in research applications, such as Western blotting, immunoprecipitation, and immunohistochemistry, to study the expression and function of the E2F1 protein.

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4 protocols using e2f1 c20

1

Western Blot Analysis of Protein Targets

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For immunoblots, cells were harvested in radioimmunoprecipitation assay buffer [50 mM tris-HCl (pH 8), 150 mM NaCl, 1% (w/v) Igepal CA-630, 0.5% (w/v) sodium deoxycholate, 0.1% (w/v) SDS, 0.2 mM sodium orthovanadate, and protease inhibitor cocktails] and incubated on ice for 30 min before SDS–polyacrylamide gel electrophoresis. The following antibodies were used in immunoblots: p100/TSN (A302-883A, Bethyl Laboratories; RRID: AB_10631268), E2F1 (C20, Santa Cruz Biotechnology; RRID: AB_631394), E2F1 (A300-766A, Bethyl Laboratories; RRID: AB_2096774), HA (16B12, Covance; RRID: AB_10063630), FLAG (M2, Sigma-Aldrich; RRID: AB_262044), β-actin (AC-74, Sigma-Aldrich; RRID: AB_476697), H4R3me2s (ab5823, Abcam; RRID: AB_10562795), histone H4 (ab10158, Abcam; RRID: AB_296888), and SENP7 (donated by R. Hay, University of Dundee, UK).
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2

ChIP Assay for E2F1 Regulation

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ChIP assay was conducted following the manufacture’s protocol (Active Motif). Briefly, after arsenic treatment, cells (5 × 107) were fixed with 1% formaldehyde at RT for 5 min, followed by nuclei extraction. ChIP assay was conducted overnight at 4 °C using 7 μg of DNA and 3 μg of control IgG or E2F1 (C-20, Santa Cruz) after enzyme digestion of chromatin at 37 °C for 30 min. We used protein G magnetic beads (Active Motif) to capture the ChIP product, which was digested with proteinase K after reversion of cross-links for 15 min at 95 °C. The isolated DNA was used as the template for PCR amplification. The primers used for Aurora-A promoter were: site 1 (−268 to −80), sense 5′-TGGTCCGGTTCTCTTGGTAT-3′ and antisense 5′-AAGCTTGACGCATTGGAGAT-3′; site 2 (−104 to +106), sense 5′-CGACGCGTTGGCTCCACCACTTCCGG-3′ and antisense 5′-CCAGGAGCTCAGCCGTTAGAATTCAAAGG-3′ (He, [22 (link)]).
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3

Antibody-Based Protein Detection

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Antibodies against the following proteins were used for FACS, immunoblotting and IF staining: MICA/B (6D4; BioLegend), mouse IgG2a isotype Ctrl (MOPC-173), Annexin V (BioLegend), pChk1 S345, Chk1, Ku80 (all from Cell Signaling Technology, Inc., Beverly, MA, USA) and E2F1 (C-20; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA).
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4

Comprehensive Protein Expression Analysis

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Sample preparation as previously described [14 (link)]. The primary antibodies at the indicated dilutions: c-Abl (K12; Santa-Cruz Cat#sc-131), β-actin (Sigma-Aldrich Cat#A5441), phospho-ATR-S428 (ABclonal Inc., USA; Cat#AP0676), phospho-chk1-S345 (ABclonal Cat#AP0578), phospho-ATM-S1981 (ABclonal Cat#AP0008), phospho-chk2-Thr68 (Cell Signaling Technology [CST] Cat#2197), GAPDH (CST Cat#5174), CCND1 (A12; Santa-Cruz Cat#sc-8396), CCNE (C19; Santa-Cruz Cat#sc-198), E2F1 (C20; Santa-Cruz Cat#sc-193), p27[Kip1] (BD Cat#610242), RB (IF8; Santa-Cruz Cat#sc-102), PARP (CST Cat#9542), Bcl-xL(H-5) (H5; Santa-Cruz Cat#SC-8392). About the phosphorylation array, the Proteome Profiler Human Phospho-MAPK Array Kit (ARY002B; R&D Systems, Minneapolis, MN) was used according to the manufacturer’s instructions.
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