MDA-MB-231 and MCF-7 cells were treated with WA, or DMSO solvent control as indicated in the figure legends. At the end of the incubation time, total cell lysates were prepared in RIPA buffer. Protein concentrations were determined by the DC protein assay (Bio-Rad, CA, USA). Equal amounts of protein were prepared in SDS-Laemli sample buffer (62.5mM Tris–HCl pH6.8, 2% SDS, 10% glycerol, 0.5% DTT). Proteins were separated on 8.5% SDS-PAGE, and transferred onto a nitrocellulose membrane. Non-specific binding sites on the membrane were blocked with a mixture of 50% Licor blocking buffer (Licor, Lincoln, NA, USA)/50% TBS containing 0.2% Tween-20 for 1 hour. Afterwards membranes were incubated with DNMT1 (MG-261,Imgenex), DNMT3A (#3598, Cell Signaling), DNMT3B (Ab16049, Abcam), JARID1B (HPA027179, Sigma Aldrich), or β-Actin (A5441, Sigma Aldrich) recognizing primary antibodies and visualized with fluorophore-coupled secondary antibody. β-Actin was used as a loading control protein. Detection was performed by use of the Odyssey Imaging System (Licor, Lincoln, NA).
Dnmt1
Manufactured by Novus Biologicals
Sourced in United States
DNMT1 is a DNA methyltransferase enzyme responsible for maintaining DNA methylation patterns during cell division. It plays a crucial role in the regulation of gene expression and genomic stability.
Automatically generated - may contain errors
Lab products found in correlation
Dnmt3a, by Novus Biologicals (4 mentions)
Dnmt3b, by Novus Biologicals (4 mentions)
Dnmt3b, by Abcam (3 mentions)
β actin, by Merck Group (2 mentions)
Znf304, by Fortis Life Sciences (2 mentions)
Setdb1, by Merck Group (2 mentions)
β actin, by Santa Cruz Biotechnology (2 mentions)
Pvdf membrane, by Merck Group (2 mentions)
Odyssey imaging system, by LI COR (1 mentions)
Blocking buffer, by LI COR (1 mentions)
Dc protein assay, by Bio-Rad (1 mentions)
Dnmt3a, by Cell Signaling Technology (1 mentions)
Cebpd sc 636, by Santa Cruz Biotechnology (1 mentions)
Img 261a, by Novus Biologicals (1 mentions)
Casp8, by Thermo Fisher Scientific (1 mentions)
α tubulin, by Merck Group (1 mentions)
Ar pg 21, by Merck Group (1 mentions)
Casp3, by Cell Signaling Technology (1 mentions)
H3k9me3, by Merck Group (1 mentions)
C jun, by Merck Group (1 mentions)
14 protocols using dnmt1
1
Western Blot Analysis of DNMT and JARID1B
2
Protein Expression and Apoptosis Assay
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The experimental cells were harvested and lysed in RIPA buffer (50 mM Tris-HCl (pH 7.4), 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.25% sodium deoxycholate, 1 mM DTT, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, aprotinin 1 μg/ml, leupeptin 1 μg/ml and 1 mM Na3VO4). The proteins were separated by SDS-PAGE, transferred to PVDF membranes and probed with specific antibodies as follows: AR (pg-21, Millipore, Billerica, MA, USA), CEBPD (SC-636, Santa Cruz, Dallas, TX, USA), DNMT1 (IMG-261A, IMGENEX, San Diego, CA, USA), DNMT3A (IMG-268A, IMGENEX), DNMT3B (IMG-184A, IMGENEX), E2F1 (SC-193, Santa Cruz), EZH2 (07-400, Upstate, Billerica, MA, USA), HA (MMS-101R, COVACE, Princeton, NJ, USA), H3K27 trimethylation, (07-449, Upstate), myc (SC-40, Santa Cruz), CASP8 (RB-1200, Thermo Scientific, Pittsburgh, PA, USA), CASP3 (9661, Cell Signaling Technology, Danvers, MA, USA), SUZ12 (07-379, Upstate), tBid (2002, Cell Signaling Technology) and α-tubulin (T6199, Sigma). Mitochondrial protein and cytosolic protein were isolated using the Cytochrome c Releasing Apoptosis Assay kit according to the manufacturer's instructions (K257-100, BioVison, San Francisco, CA, USA).
3
Chromatin Immunoprecipitation Assay Protocol
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ChIP assays were performed as previously described (Gazin et al., 2007 (link)) using the following antibodies: ZNF304 (described above), KAP1 (Bethyl Laboratories), SETDB1 (Millipore, Billerica, MA), DNMT1, DNMT3A, and DNMT3B (all from Imgenex, San Deigo, CA), cJUN (Millipore), H3K27me3 (Cell Signaling Technology), H3K9me3 (Millipore), H3K4me3 (Abcam), EZH2 (Millipore) and BMI1 (Abcam). The CDX1 antibody (Chan et al., 2009 (link)) was kindly provided by Walter Bodmer (University of Oxford, UK). ChIP products were analyzed by qRT-PCR (see Supplementary file 1 for primers). Samples were quantified as percentage of input, and then normalized to an irrelevant region in the genome (∼3.2 kb upstream from the transcription start site of GCLC). Fold enrichment was calculated by setting the IgG control IP sample to a value of 1.
4
ChIP Assay Protocol with Antibodies
5
Comprehensive Antibody Validation Protocol
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References of primary antibodies used in this study are: ZBTB38 (homemade antibody #148), USP9X (Millipore #MABE352; Abcam #19879; Bethyl Lab #A301-351A), GST (CST #2622), GAPDH (Abcam #ab9485), USP7 (Abcam #4080), HA (CST,#2367), H2AX (Bethyl Lab #A300-083A), phospho-H2AX (Millipore #05–636), TUBULIN (Abcam #ab7291), RFP (Rockland #600-401-379), V5 (Santa Cruz #sc-84594), MYC (CST #2276), WEE1 (Abcam #79298), NRF2 (Abcam #ab62352), PGC1 (Abcam #72230), DNMT1 (Imgenex #IMG-261A), ORC2 (Santa Cruz #28742), phospho-CHK1 (CST #2341), phospho-CHK2 (CST #2661), phospho-ATM (CST #4526P), PARP1 (CST #9542), JNK (Santa Cruz #474) and 53BP1 (Abcam #21083).
6
Profiling APC Protein Expression
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Flow-sorted CD34+, CD29+, Sca-1+ primary APCs and EVC005 cells (treated with or without 1DSO) were homogenized in RIPA buffer with protease and phosphatase inhibitors. Lysates from primary APCs were run on ProteinSimple JESS instrument for UCP2 (Cell Signaling, 89326), CEPBa (Cell Signaling, 8178), NR2F2 (Cell Signaling, 6434) and DNMT1 (Novus Biologicals, NB100-56519) antibodies. The antibodies used while running the EVC005 lysate were TOM20 (Cell Signaling, 42406), SOD2 (Cell Signaling, 13141), NRF1 (Cell Signaling 46743), HSP90 (Cell Signaling, 4874), OxPhos Rodent WB Antibody cocktail (ThermoFisher, 45–8099) and GAPDH (Cell Signaling, 5174). All antibodies were used at a dilution of 1/50 for JESS westerns. Three microliters of 1.2 mg/mL protein was loaded per well for the JESS assays. Proteins from primary APCs from 6 different SVFs for each condition (high or low n6/n3) were used for each JESS assay.
7
Comprehensive Analysis of Epigenetic Regulators
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Antibodies specific for EP2, EP3, EP4, Sp1, Sp3, TET2, TET3, β-actin, vinculin, and histone H3 were purchased from Santa Cruz Biotechnology (Dallas, TX). The following antibodies were purchased: COX-2 and 5-methylcytosine (5mC) from Cell Signaling (Danvers, MA); EP1 from Abcam (Cambridge, MA); Dnmt1, Dnmt3a, and Dnmt3b from Novus Biologicals (Littleton, CO); and TET1 from Epigentek, Inc (New York, NY). The Methylamp Global DNA Methylation Quantification Kit, the EpiQuik DNA Methyltransferase Activity Assay Kit, and TET Assay Activity Kit (Epigenase 5mC-hydroxylase TET activity/inhibition assay kit) were purchased from Epigentek, Inc. (New York, NY). The PGE2 immunoassay kit was purchased from Cayman Chemical (Ann Arbor, MI). All other chemicals of analytical grade were purchased from Sigma-Aldrich Chemical Co (St Louis, MO). Purified honokiol was purchased from Quality Phytochemicals, LLC (Edison, NJ).
8
Formalin-Fixed Lung Tissue Preparation for IHC
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The superior and inferior lobe of the left lung was inflated with 1% neutral buffered formalin in 0.5% agarose at 20 cm H2O pressure and fixed in 10% neutral buffered formalin as described previously (6 (link)). The fixed lung was blocked and embedded in paraffin, after which 5-μm sections were cut and probed for DNMT1 (Novus), DNMT3A (Epigentek), and DNMT3B (Thermo Fisher) by immunohistochemistry and images were acquired on Nikon Eclipse 90-i microscope.
9
Western Blot Antibody Panel
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The following primary antibodies were used for western blotting mouse anti-actin (A4700, Sigma Aldrich), PCNA (PC10, Millipore), rabbit anti-Cdt1 (D10F11, Cell Signalling Technology), DNMT1 (NB100-56519, Novus Biologicals), Fen1 (EPR4459(2), GeneTex), Ligase I (PA5-27820, Thermo Scientific), p21 (EPR362, abcam), Polη (ab17725, abcam), Ubiquitinated-PCNA (D5C7P, Cell Signalling Technology).
10
Analyzing Spleen Protein Signaling Cascades
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Mice spleens were homogenized using RIPA containing a protease and phosphatase inhibitor (Roche). Nuclear proteins and cytoplasmic proteins were extracted from mice spleens using NE-PER™ Nuclear and Cytoplasmic Extraction Reagents (Thermo). Protein content was quantified using a Pierce™ BCA Protein Assay Kit (Thermo) and proteins were separated via SDS-PAGE with either 12.5%, 10% or 7.5% acrylamide and transferred into PVDF membranes (Millipore, Burlington, MA, USA). Next, blots were blocked with 5% milk or 5% BSA and probed with specific primary antibodies against phosphor-NF-κb p65, NF-κb p65, phosphor-IκBα, IκBα, phosphor-ERK, ERK (Cell Signaling Danvers, MA, USA), STK3/MST-2, phosphor- MST1/ MST2 (Abcam, Cambridge, UK), Phosphor-YAP, YAP, VEGF Receptor 2, Phosphor-VEGF Receptor 2, Phosphor-EGF Receptor, EGF Receptor (Cell Signaling, Danvers, MA, USA), DNMT1(Novus, Centennial, CO, USA), DNMT3a and DNMT3b (Abcam, Cambridge, UK) overnight at 4°C. The membranes were then washed with TBST and incubated with horseradish peroxidase-conjugated secondary antibodies at room temperature for 1 h. Next, target proteins were detected using chemiluminescence via Bio-rad ChemiDoc MP and normalized to β-actin or histone H3.
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