First, 1% formaldehyde was added to cross-link the cells, and 10 min later, glycine was used to stop the reaction. To shear genomic DNA from the cross-linked cells, micrococcal nuclease was used to lyse and enzymatically digest the cells. An anti-c-Jun antibody (Abcam) was used for immunoprecipitation, with IgG serving as a negative control. After the protein/DNA complexes were eluted from the beads, they were treated with proteinase K solution for 2.5 h at 65 °C and then analysed by real-time PCR and semiquantitative PCR using primers for the ESR2 promoter. The specific primers used to amplify target sequences from human ERβ promoters are listed in Supplementary Table S3 .
Anti c jun antibody
Manufactured by Abcam
Sourced in United States
Anti-c-Jun antibody is a primary antibody that recognizes the c-Jun protein. c-Jun is a transcription factor that is a part of the AP-1 complex, which regulates gene expression in response to various stimuli.
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Lab products found in correlation
Anti rabbit igg alexa fluor 488 conjugate, by Cell Signaling Technology (1 mentions)
Anti nf κb p65 antibody, by Cell Signaling Technology (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Fluoview fv1000, by Olympus (1 mentions)
Easy load pcr master mix, by Beyotime (1 mentions)
Mouse control igg, by Beyotime (1 mentions)
Chip assay kit, by Beyotime (1 mentions)
Anti mct4 antibody, by Abcam (1 mentions)
Chemiluminescence system, by Bio-Rad (1 mentions)
Anti glut4 antibody, by Abcam (1 mentions)
Anti pdk1 antibody, by Abcam (1 mentions)
Anti glut1 antibody, by Abcam (1 mentions)
Anti pdk4 antibody, by Abcam (1 mentions)
Anti c myc antibody, by Abcam (1 mentions)
Anti hif 1 antibody, by Abcam (1 mentions)
Anti junb antibody, by Abcam (1 mentions)
Anti ubiquitin antibody, by Abcam (1 mentions)
Anti mdm2 antibody, by Abcam (1 mentions)
Anti elk1 antibody, by Abcam (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
5 protocols using anti c jun antibody
1
ChIP-qPCR Assay for Estrogen Receptor β
2
Immunofluorescence Analysis of NF-κB and c-Jun Activation
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RAW 264.7 cells were grown on coverslips and treated with FA-97 (1 μM) for 24 h, followed by stimulation with LPS (1 μg/ml) for another 2 h. Cells on coverslips were fixed with 4% paraformaldehyde, permeabilized in 0.2% Triton X-100 and incubated with 3% BSA. After incubated with rabbit monoclonal anti-NF-κB (p65) antibody (1:100, Cell Signaling Technology, Danvers, MA, USA) or anti-c-Jun antibody (1:100, Abcam, Inc., Cambridge, UK) in a humidified chamber overnight at 4°C, cells were exposed to Alexa Fluor® 488 conjugate anti-rabbit IgG (1:2000, Cell Signaling Technology, Danvers, MA, USA) for 30 min at room temperature and stained with DAPI (1:1000, Invitrogen, Carlsbad, CA, USA). Cells were observed and photographed with a confocal laser scanning microscope (Fluoview FV 1000, Olympus, Tokyo, Japan).
3
ChIP Assay for β3GnT8 Promoter
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ChIP was performed using a ChIP assay kit (Beyotime, Haimen, China) according to the manufacturer's protocol. Chromatin solutions were sonicated and incubated with the anti-c-Jun antibody (Abcam, Cambridge, MA, USA) or mouse control IgG (Beyotime, Haimen, China) while rotating overnight at 4°C. The solution was washed according to the manufacturer's instruction. DNA-protein cross-links were reversed, and chromatin DNA was purified and subjected to PCR analysis using the Easy-Load PCR Master mix (Beyotime, Haimen, China). Primers 5’-TGTACGCGTGAGGCACATGGCAAAGG-3’ (forward) and 5’-GTTCTCGAGAGTGGGGAGGAAGTGGT-3’ (reverse) were used to amplify the β3GnT8 promoter sequence [19 (link)]. Following amplification, PCR products were resolved on a 1.5% agarose gel and visualized by ethidium bromide staining.
4
Western Blot Analysis of Cellular Proteins
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Following treatment or transfection, culture supernatants were removed, cells or tissues were lysed using radio immunoprecipitation assay buffer (RIPA, Beyotime, China), and protein was harvested and quantified by bicinchoninic acid (BCA) analysis (Beyotime, China). Protein extracts were separated in 10% SDS-PAGE gels and transferred onto polyvinylidene fluoride (PVDF) membranes (Sigma-Aldrich, USA). After incubation with a high affinity anti-c-Myc antibody (1:1000, Abcam), anti-ELK1 antibody (1:1000, Abcam), anti-cdc25c antibody (1:1000, Abcam), anti-hif1 antibody (1:1000, Abcam), anti-c-Jun antibody (1:1000, Abcam), anti-Mdm2 antibody (1:1000, Abcam), anti-JunB antibody (1:1000, Abcam), anti-mct4 antibody (1:1000, Abcam), anti-pdk1 antibody (1:1000, Abcam), anti-pdk4 antibody (1:1000, Abcam), anti-GLUT1 antibody (1:1000, Abcam), anti-GLUT4 antibody (1:1000, Abcam), anti-Ubiquitin antibody (1:1000, Abcam) or anti-β-actin antibody (1:2000, Cell Signaling Technology, USA) in Primary Antibody Dilution (MB9881, Dalian Meilun Biotechnology Co., Ltd), the membranes were incubated with a secondary antibody (1:5000, Cell Signaling Technology, USA). After washes, signals were detected using FDbio-Femto ECL (Fudebio, Hangzhou, China) and a chemiluminescence system (Bio-Rad, USA). All images were analyzed using Image Lab software.
5
Immunohistochemical Evaluation of Transcription Factors in Lung and Colon
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IHC assay was carried out to assess activating transcription factor 2 (ATF2), c-jun, and c-fos expressions in lung and colon tissues. Briefly, 6 µm lung or colon sections were de-paraffinized in xylene solution and hydrated in a decreasing gradient ethanol solution. Sections were then treated with citrate antigen retrieval solution (Beyotime Biotechnology, 20 min, 95 ℃), permeabilized using 0.5% Triton X-100 solution (20 min), incubated with 3% H2O2 solution, and coated with 3% bovine saline albumin (BSA, Beyotime Biotechnology). Sections were then treated with anti-ATF2 antibody (1:250), anti-c-jun antibody (1:250), and anti-c-fos antibody (1:250, Abcam Biotechnology, MA, USA) overnight at 4 ℃ and HRP-conjugated secondary antibody for 60 min. Relative expressions were visualized using a diaminobenzidine (DAB) solution (Beyotime Biotechnology) and the cell nucleus was stained using hematoxylin solution. Results were observed under a microscope (Nikon, Japan).
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