ChIP assay was undertaken using EZ‐ChIP kit (Upstate Biotechnology, Temecula, CA) (Zhao et al, 2016 ; Li et al, 2018b ), with antibodies (1:100 dilution) specific for CUX1 (#81557, Cell Signaling Technology, Inc., Danvers, MA) or MAZ (ab85725, Abcam Inc.) and primers targeting gene promoters (Appendix Table S1 ).
Ab85725
Manufactured by Abcam
Ab85725 is a laboratory equipment product. It is a device used for a specific function in scientific research and experimentation. No further details about its core function or intended use can be provided in an unbiased and factual manner.
Automatically generated - may contain errors
Lab products found in correlation
Ab9106, by Abcam (1 mentions)
Ab18230, by Abcam (1 mentions)
Ab8580, by Abcam (1 mentions)
Ab4729, by Abcam (1 mentions)
Ab220788, by Abcam (1 mentions)
Ab4174, by Abcam (1 mentions)
C15410195, by Diagenode (1 mentions)
Ab18785, by Abcam (1 mentions)
Ripa buffer, by Thermo Fisher Scientific (1 mentions)
Tgx gel, by Bio-Rad (1 mentions)
0.2 m pvdf membrane, by Bio-Rad (1 mentions)
Ab38449, by Abcam (1 mentions)
Ab76302, by Abcam (1 mentions)
Ab205718, by Abcam (1 mentions)
Ab9485, by Abcam (1 mentions)
Ab32536, by Abcam (1 mentions)
6 protocols using ab85725
1
ChIP Assay for CUX1 and MAZ
2
ChIP-qPCR Assay for MAZ Binding
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In a 10 cm dish, 1% formaldehyde solution was added to TNBC cells (1.5 × 107) for 10 min at room temperature. Then, the cells were washed three times with PBS and harvested in chromatin immunoprecipitation (ChIP) lysis buffer supplemented with protease inhibitor. After the cells were lysed for 20 min, cells were sonicated on ice to shear the DNA into 0.1–0.5 kb fragments. The supernatant was centrifuged at 12,000 rpm for 15 min. Immunoprecipitation was then performed by incubating with anti-MAZ antibody (Abcam, ab85725) overnight at 4°C. Then, 60 μl of protein G agarose was added to the DNA–protein complexes overnight, and the associated genomic DNA was amplified by real-time PCR.
3
EWSR1 and MAZ Co-IP Analysis
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4
Antibodies for Chromatin Immunoprecipitation
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Custom-made mouse monoclonal CTCF antibodies were previously described. [96 (link)]. Custom-made mouse monoclonal BORIS antibodies were also described previously [22 (link)]. Rabbit polyclonal antibody against SRCAP (Kerafast, ESL103), rabbit polyclonal against HCFC1 (Novus Biologicals, NB100-68209), goat affinity-purified polyclonal antibody against Mxi1 (R&D Systems, AF4185), rabbit monoclonal to TATA-binding protein TBP (Abcam, ab220788), rabbit polyclonal anti-PHF8 antibody (Bethyl Laboratories, A301-772A), rabbit polyclonal to MAZ (Abcam, ab85725), rabbit polyclonal antibody against the region of histone H3 containing the trimethylated lysine 27 (H3K27me3) (Diagenode, C15410195), rabbit polyclonal antibody against histone H3, trimethylated at lysine 36 (H3K36me3) (Diagenode, C15410058), rabbit polyclonal antibody against histone H2A.Z (Abcam, ab4174), rabbit polyclonal to Histone H3 (acetyl K27) (Abcam, ab4729), rabbit polyclonal to Histone H3 (tri methyl K4) (Abcam, ab8580), anti-RNA Polymerase II Antibody, CTD Antibody, clone 8WG16 (Millipore, 05–952-I-100UG).
5
Western Blot Analysis of Cellular Proteins
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RIPA buffer (Thermo Scientific) containing Complete EDTA-free protease inhibitor was used to isolate proteins from cells and tissues. Proteins (30 μg) were loaded with a 4–15% TGX gel (BioRad) and then transferred to 0.2 µm PVDF membranes (BioRad). The membranes were blocked in 5% non-fat dried milk/Tris-buffered saline and 0.1% Tween-20 for 1 h at room temperature. Next, the membranes were incubated with primary antibodies: anti-SIPL1 (Abcam, ab79039), anti-MAZ (Abcam, ab85725), anti-AKT (Abcam, ab18785), anti-p-AKT (Abcam, ab38449), anti-P65 (Abcam, ab32536) or anti-p-P65 (Abcam, ab76302), and anti-GAPDH (Abcam, ab9485) overnight, along with horseradish peroxidase-labeled IgG (Abcam, ab205718) at room temperature. The protein bands were detected using enhanced chemiluminescence (Pierce, Rockford, IL, USA). Densitometry analysis was performed using ImageJ software (version 1.36; National Institutes of Health, Bethesda, MD, USA).
6
Characterization of TBK1 Enrichment
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293T cells were fixed with 16% methanol, cross‐linked, lysed, sonicated, and then cultured with MAZ antibody (ab85725; Abcam) overnight. Beads were used to collect the protein–DNA complex. NaCl was added for cross‐linking. The enrichment of TBK1 was examined using RT‐qPCR.
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