MCF-7 cells were cultured in 10% charcoal-stripped serum containing medium for 3 days and then treated with 10 nM or 100 nM estradiol for 0, 15, 30, 45 and 60 minutes. Cells were then fixed in 1% formaldehyde and subjected to Chromatin immunoprecipitation using anti-ERα, anti-SRC-3, anti-CARM1 (Invitrogen), anti-H3K18ac (Active Motif) specific antibodies following the manufacturer’s protocol (Active motif). Binding of these proteins to TFF1 gene promoter was assessed through real time PCR using the following primers. Forward: 5′-GGATTAAGGTCAGGTTGGAGG; Reverse: 5′-CATGGTCAAGCTACATGGAAGG. TFF1 ERE3 (−9.9 kb from the TSS); Forward: 5′-GTCGTTGCCAGCGTTTCC Reverse: 5′-CTTCTCCACGCCCTGTAAATTT; GREB1 ERE1 (−1.6 kb from isoform a TSS): Forward: 5′-GTGGCAACTGGGTCATTCTGA; Reverse: 5′-CGACCCACAGAAATGAAAAGG. GREB1 distal enhancer (−35.4 kb from isoform a TSS) Forward: 5′-CAGGGGCTGACAACTGAAAT; Reverse: 5′-GAGAGGGTGGTGACACTTGG.
Anti h3k18ac
Manufactured by Active Motif
Sourced in Belgium, United States
Anti-H3K18ac is a laboratory product that detects the acetylation of lysine 18 on histone H3. This modification is associated with active transcription. The product is designed for use in various research applications, such as chromatin immunoprecipitation (ChIP) and Western blotting.
Automatically generated - may contain errors
Lab products found in correlation
Lightcycler, by Roche (2 mentions)
Anti rnapol 2, by Cell Signaling Technology (2 mentions)
Ab5070, by Abcam (1 mentions)
Anti flag m2 peroxidase, by Merck Group (1 mentions)
Anti h3k27ac, by Active Motif (1 mentions)
Anti gfp antibody, by Takara Bio (1 mentions)
Anti mouse igg hrp, by Bio-Rad (1 mentions)
Anti h4k12ac, by Active Motif (1 mentions)
Chemi lumi one, by Nacalai Tesque (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
Chemidoc mp imaging system, by Bio-Rad (1 mentions)
Ab992, by Abcam (1 mentions)
Ab4729, by Abcam (1 mentions)
Ab32146, by Abcam (1 mentions)
Ab125096, by Abcam (1 mentions)
Ab18184, by Abcam (1 mentions)
Anti h3, by Active Motif (1 mentions)
Anti pr h 190 sc 7208, by Santa Cruz Biotechnology (1 mentions)
Anti ctcf, by Merck Group (1 mentions)
Anti h3k27ac, by Abcam (1 mentions)
6 protocols using anti h3k18ac
1
Estrogen Receptor Dynamics in MCF-7 Cells
2
Western Blot Analysis of RNAi Pathway Proteins
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Protein samples were denatured by boiling at 95°C for 3 min in protein-loading buffer [2% (w/v) SDS, 100 mM DTT, 0.05% (v/v) BPB, and 10% (v/v) glycerol], resolved by SDS-PAGE, and transferred to 0.2-μm polyvinylidene difluoride membrane (Wako) using the semi-dry system (Trans-blot Turbo, Bio-Rad). The membrane was blocked in 4% (w/v) skim milk (Nacalai) in 1× phosphate-buffered saline (PBS) supplemented with 0.1% (v/v) Tween 20 and further incubated with the following antibodies: anti-Aub antibody (1:500; guinea pig) (80 (link)), anti-GFP antibody (1:2000; Clontech, rabbit), anti-Ago1 antibody (1:1000; Abcam, Ab5070, rabbit), anti-Ago2 antibody (1:100; guinea pig) (25 (link)), anti-Piwi (1:10; mouse, P4D2), anti-FLAG M2-peroxidase [horseradish peroxidase (HRP)] (1:5000; Sigma-Aldrich, #A8592), anti-H3K18Ac (1:2000; Active Motif, #39756), anti-H3K27Ac (1:2000; Active Motif, #39136), anti-H4K8Ac (1:2000; Active Motif, #61104), anti-H4K12Ac (1:2000; Active Motif, #39928), anti-guinea pig immunoglobulins-HRP (1:1000; Dako), anti-rabbit immunoglobulin G (IgG)–HRP (1:3000; Bio-Rad), anti-mouse IgG-HRP (1:3000; Bio-Rad). The chemiluminescent signals were obtained by using Chemi-Lumi One (Nacalai) and detected by Chemidoc MP Imaging system (Bio-Rad). The images were processed by using Pixelmator or Fiji. The immunoblot signals were quantified by using Fiji.
3
Quantitative Chromatin Immunoprecipitation Analysis
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Chromatin immunoprecipitation (ChIP) assays were performed as described (12 (link)) using anti-PR (H190 SC-7208, Santa Cruz), anti-ERK2(D12) (05-157, Merck), anti-SRC3-(5E11) (#2126, Cell Signaling), anti-p300, clone NM11 (#61401, Active Motif), anti-RNApol II (#2629, Cell Signaling), anti-RAD21 (ab992, Abcam), anti-CTCF (07-729, Merck), anti-H3K27ac (ab4729, Abcam), anti-H3K18ac (#39693, Active Motif) and anti-BRD4 (kind gift from Cheng-Ming Chiang, UT Southwestern Medical Center). Quantification of ChIP was performed by real-time PCR using Roche LightCycler (Roche). The fold enrichment of target sequence in the immunoprecipitated (IP) compared to input (Ref) fractions was calculated using the comparative Ct (the number of cycles required to reach a threshold concentration) method with the equation 2Ct(IP) − Ct(Ref). Each of these values was corrected by the human β-globin gene and referred to as relative abundance over time zero. Primer sequences for HAs, medium accessible sites (MAs) and low accessible sites (LAs) are available in Supplementary Table S1 .
4
Immunoblot Analysis of Protein Expression
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The fresh hyphae of tested strains were collected, and the proteins were isolated using protein lysis buffer [50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 5 mM EDTA, and 1% Triton X-100]. After quantification, equal amounts of protein from different samples were loaded onto the 12% SDS-PAGE gel. After electrophoresis and membrane transfer, anti-GFP (ab32146; Abcam, Cambridge, UK), anti-mCherry (ab125096, Abcam), anti-His antibody (ab18184, Abcam), anti-H3 (no. 61475; Active Motif, La Hulpe, Belgium), anti-H3K18ac (no. 39756, Active Motif), anti-GST (M0807; Hua'an, Hangzhou, China), anti-GAPDH antibody (EM1101, Hua'an), and anti-Ub (ET1609-21, Hua'an) antibodies were used for immunoblot analyses.
5
Histone Acetylation Assay with FgGCN5 and FgPacC30
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An in vitro histone acetylation assay was carried out following a previous publication with minor modifications (51 (link)). Briefly, purified histone proteins (H3-His and H2B-His) was incubated with purified FgGCN5-His protein in HAT buffer (50 mM pH 8.0 Tris–HCl, 50 mM KCl, 0.1 mM EDTA, 1 mM DTT, 1 mM protease inhibitor, 5% glycerol, and 5 mM acetyl-CoA) in the absence or presence of increasing concentrations of FgPacC30-His protein. The resulting reactions were then resolved by SDS-PAGE and analyzed by western blotting using anti-H3K18ac (39755, Active Motif Inc., Carlsbad, USA), anti-H2BK11ac (ab240613, Abcam, Cambridge, UK) antibodies.
6
Chromatin Immunoprecipitation (ChIP) Assay
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ChIP assays were performed as described (Strutt and Paro, 1999 (link)) using anti-PR (H190 SC-7208, Santa Cruz,); anti-RNApol II (#2629, Cell Signaling); anti-CTCF (07-729, Merck); anti-H3K27ac ( ab4729, Abcam); anti-H3K18ac (#39693, Active Motif). Quantification of chromatin immunoprecipitation was performed by real-time PCR using Roche Lightcycler (Roche). The fold enrichment of target sequence in the immunoprecipitated (IP) compared to input (Ref) fractions was calculated using the comparative Ct (the number of cycles required to reach a threshold concentration) method with the Eq. (2) Ct(IP)-Ct(Ref). Each of these values was corrected by the human β-globin gene and referred as relative abundance over time zero. Primers sequences for target regions are available on request.
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