The promoter region of FOSB gene was amplified from the genomic DNA of 293T cells and inserted into pGL4.15 vector (Promega, Madison, Wisconsin, USA). For the luciferase reporter assays, HEK293T cells were seeded in 24-well plates and transfected with the indicated plasmids using Lipo2000 for 36 h. Luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega). The firefly luciferase luminescence data were normalized by the Renilla luciferase luminescence data. A chromatin immunoprecipitation assay kit was used (Millipore, USA). In brief, cells fixed with 1% formaldehyde (Sigma, USA) and harvested in SDS lysis buffer. DNA was sheared to fragments of 200–1000 bp by sonications. Lysates containing soluble chromatin were incubated and precipitated overnight with 2 μg of anti- histone H3K27me3 (ab6002, Abcam), anti-histone H3K27ac (#8173, Cell Signaling), anti-EZH2 (E7031, Sigma-Aldrich); anti-C/EBPβ (sc-7962, Santa Cruz) or rabbit IgG (#ab172730, Abcam). Protein G agarose was then added for 4 h. Protein-DNA crosslinks were reversed by treatment with proteinase K for 2 h at 45 °C. The DNA was subsequently purified, diluted and subjected in the quantitative real-time PCR reactions.
Anti histone h3k27ac
Manufactured by Cell Signaling Technology
Anti-histone H3K27ac is a laboratory reagent used to detect the presence and quantity of acetylated lysine 27 on histone H3 protein. It is a specific antibody that binds to this post-translational modification, allowing researchers to study chromatin structure and gene regulation processes.
Automatically generated - may contain errors
Lab products found in correlation
Ab6002, by Abcam (2 mentions)
Ab172730, by Abcam (2 mentions)
Chromatin immunoprecipitation assay kit, by Merck Group (2 mentions)
Pgl4.15 vector, by Promega (2 mentions)
Anti ezh2, by Merck Group (2 mentions)
Anti c ebpβ sc 7962, by Santa Cruz Biotechnology (2 mentions)
Formaldehyde, by Merck Group (2 mentions)
Dual luciferase reporter assay system, by Promega (2 mentions)
Anti histone h3k27me3, by Abcam (2 mentions)
Anti gapdh sc 47724, by Santa Cruz Biotechnology (1 mentions)
Anti fos b f 7, by Santa Cruz Biotechnology (1 mentions)
Anti cebpb, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
4 protocols using anti histone h3k27ac
1
FOSB Gene Promoter Analysis
2
Western Blot Analysis of Epigenetic Markers
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Cells were lysed with lysis buffer (100 mM Tris–HCl, pH 6.8, 100 mM DTT, 1% SDS, 10% glycerol). Proteins were separated by 10–12% SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk in phosphate-buffered saline (PBS) for 1 h before incubation with primary antibody overnight at 4 °C. Membranes were washed and blocked with 5% milk and incubated with different primary antibodies overnight at 4 °C, followed by incubation with secondary antibodies. The primary antibodies used in western blotting included anti-Fos B(F-7) (sc-398595; Santa Cruz; 1:1000 dilution), anti-CEBPB (sc-7962; Santa Cruz; 1:1000 dilution), anti- histone H3K27me3 (ab6002, Abcam; 1:1000 dilution), anti-histone H3K27ac (#8173, Cell Signaling; 1:1000 dilution) and anti-GAPDH (sc-47724; Santa Cruz; 1:5000).
3
Characterization of FOSB Promoter Activity
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The promoter region of FOSB gene was ampli ed from the genomic DNA of 293T cells and inserted into pGL4.15 vector (Promega, Madison, Wisconsin, USA). For the luciferase reporter assays, HEK293T cells were seeded in 24-well plates and transfected with the indicated plasmids using Lipo2000 for 36 hours.
Luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega). The re y luciferase luminescence data were normalized by the Renilla luciferase luminescence data. A chromatin immunoprecipitation assay kit was used (Millipore, USA). In brief, cells xed with 1% formaldehyde (Sigma, USA) and harvested in SDS lysis buffer. DNA was sheared to fragments of 200-1000 bp by sonications. Lysates containing soluble chromatin were incubated and precipitated overnight with 2 ug of anti-histone H3K27me3 (ab6002, Abcam), anti-histone H3K27ac (#8173, Cell Signaling), anti-EZH2 (E7031,Sigma-Aldrich); anti-C/EBPβ (sc-7962, Santa Cruz) or rabbit IgG (#ab172730, Abcam). Protein G agarose was then added for four hours. Protein-DNA crosslinks were reversed by treatment with proteinase K for 2 hours at 45°C. The DNA was subsequently puri ed, diluted and subjected in the quantitative real-time PCR reactions.
Luciferase activity was measured using the Dual Luciferase Reporter Assay System (Promega). The re y luciferase luminescence data were normalized by the Renilla luciferase luminescence data. A chromatin immunoprecipitation assay kit was used (Millipore, USA). In brief, cells xed with 1% formaldehyde (Sigma, USA) and harvested in SDS lysis buffer. DNA was sheared to fragments of 200-1000 bp by sonications. Lysates containing soluble chromatin were incubated and precipitated overnight with 2 ug of anti-histone H3K27me3 (ab6002, Abcam), anti-histone H3K27ac (#8173, Cell Signaling), anti-EZH2 (E7031,Sigma-Aldrich); anti-C/EBPβ (sc-7962, Santa Cruz) or rabbit IgG (#ab172730, Abcam). Protein G agarose was then added for four hours. Protein-DNA crosslinks were reversed by treatment with proteinase K for 2 hours at 45°C. The DNA was subsequently puri ed, diluted and subjected in the quantitative real-time PCR reactions.
4
Western Blot Analysis of Transcription Factors
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cells were lysed with lysis buffer (100 mM Tris-HCl, pH 6.8, 100 mM DTT, 1% SDS, 10 % glycerol). Proteins were separated by 10-12% SDS-PAGE, and transferred to PVDF membranes. Membranes were blocked in 5% non-fat milk in phosphate-buffered saline (PBS) for 1 h before incubation with primary antibody overnight at 4 °C. Membranes were washed and blocked with 5% milk and incubated with different primary antibodies overnight at 4℃, followed by incubation with secondary antibodies. The primary antibodies used in western blotting included anti-Fos B(F-7) (sc-398595; Santa Cruz; 1:1000 dilution), anti-CEBPB (sc-7962; Santa Cruz; 1:1000 dilution), anti-histone H3K27me3 (ab6002, Abcam; 1:1000 dilution), anti-histone H3K27ac (#8173, Cell Signaling; 1:1000 dilution) and anti-GAPDH (sc-47724; Santa Cruz; 1:5000).
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