NK92 cells or U937 cells were lysed using Laemmli lysis buffer (Sigma-Aldrich). Blots were prepared and then blocked with 5% dry milk solution in TBST for 1 h. Primary antibodies to HCA1, HAC2, HCA3, gasdermin-D, DNMT3A, DNMT3B or Caspase-1 (Abcam, Cambridge, UK) were used. HRP conjugated goat anti-rabbit or goat anti-mouse secondary antibodies (Cell Signaling Technology, Danvers, MA, USA), were diluted in fresh 5% dry milk in TBST solution and incubated with the blots for 1 h at room temperature. HRP was detected using BioRad ECL Western blotting detection reagent (BioRad, Hercules, CA, USA). Primary antibody for Actin (Cell Signaling Technology) was used to confirm loading equality.
Dnmt3b
Manufactured by Abcam
Sourced in United States, United Kingdom, China
DNMT3B is a DNA methyltransferase enzyme that catalyzes the addition of methyl groups to cytosine residues in DNA, playing a critical role in the regulation of gene expression and genomic imprinting.
Automatically generated - may contain errors
Lab products found in correlation
Dnmt3a, by Abcam (32 mentions)
Dnmt1, by Abcam (20 mentions)
Gapdh, by Abcam (6 mentions)
β actin, by Abcam (6 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (5 mentions)
Dnmt3a, by Cell Signaling Technology (4 mentions)
Dnmt1, by Cell Signaling Technology (4 mentions)
Dnmt1, by Santa Cruz Biotechnology (4 mentions)
Pvdf membrane, by Merck Group (4 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Bcl 2, by Abcam (3 mentions)
Bcl2 associated x (bax), by Abcam (3 mentions)
Ab2851, by Abcam (3 mentions)
Dnmt1, by Novus Biologicals (3 mentions)
Axio observer a1, by Zeiss (3 mentions)
Polyvinylidene fluoride membrane, by Merck Group (2 mentions)
Horseradish peroxidase coupled secondary antibody, by Beyotime (2 mentions)
Vimentin, by Abcam (2 mentions)
E cadherin, by Abcam (2 mentions)
N cadherin, by Abcam (2 mentions)
63 protocols using dnmt3b
1
Western Blot Analysis of Apoptosis-Related Proteins
2
Western Blot Analysis of Mouse Tissue Proteins
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We homogenized mouse tissues in buffer containing 50 mM Tris–HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P‐40, 0.5% deoxycholate, 0.1% SDS, and 1 mM 2‐mercaptoethanol with Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, NJ, USA) and then centrifuged them at 2,500 × g for 15 min. Equal amounts of protein were separated by 5–20% SDS–PAGE and transferred to Hybond‐P membranes (GE Healthcare, Piscataway, NJ, USA). The primary antibodies and their dilutions were as follows: AR (N20, 1:1,000; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Dnmt1 (1:1,000; Abcam, Cambridge, MA, USA), Dnmt3a (1:1,000; Abcam), Dnmt3b (1:1,000; Abcam), Hes5 (1:1,000; Santa Cruz), and ChAT (1:1,000; Millipore, Billerica, MA, USA). Primary antibody binding was probed with horseradish peroxidase‐conjugated secondary antibodies at a dilution of 1:5,000, and bands were detected by using an immunoreaction enhancing solution (Can Get Signal; Toyobo, Osaka, Japan) and enhanced chemiluminescence (ECL Prime; GE Healthcare). An LAS‐3000 imaging system (Fujifilm, Tokyo, Japan) was used to produce digital images. The signal intensities of these independent blots were quantified using IMAGE GAUGE software version 4.22 (Fuji) and expressed in arbitrary units (n = 3 for each group). The membranes were reprobed, or the same samples were examined with an anti‐GAPDH (1:5,000; Santa Cruz) antibody for normalization.
3
Western Blotting of EMT Markers
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Western blotting was performed according to standard procedures using enhanced chemiluminescence detection. Briefly, total proteins were extracted from cells using RIPA lysis buffer (Thermo Fisher Scientific). Equal amounts of protein were separated on sodium dodecyl sulfate–polyacrylamide gels and then polyvinylidene fluoride membrane (Millipore). After blocking with 5% skim milk, the membranes were incubated with the primary antibodies for DNMT3A, DNMT3B, E-cadherin, N-cadherin, vimentin and GAPDH (Abcam), followed by incubation with corresponding horseradish peroxidase-coupled secondary antibody (Beyotime). The signals were detected with enhanced chemiluminescence reagents (Pierce). GAPDH was used as an internal control.
4
Western Blot Analysis of DNMT and JARID1B
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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).
5
Protein Expression Analysis by Western Blot
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Western blots performed with 10, 12, or 15% SDS-polyacrylamide gels were incubated with primary antibodies for IL-1ß (R&D Systems, Minneapolis, MN), NLRP3 (LSBio, Seattle, WA), Ogg1 (Novus Biologicals, Littleton, CO), Caspase 1 (Genetex, Irvine, CA), and DNMT3b (Abcam, Cambridge, MA). Western blots were visualized using alkaline phosphatase-conjugated secondary antibodies (Sigma-Aldrich).
6
Immunoblotting of Dopaminergic Markers
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This was performed as previously described [20 (link)] with antibodies against D2R (1:500, rabbit), dopamine transporter (DAT; 1:500, rabbit), tyrosine hydroxylase (TH; 1:1000, rabbit) (AB5084P, AB1591P and AB152, Merck Millipore, Billerica, MA, USA), signal transducer and activator of transcription 3α (STAT3α; 1:1000, rabbit), DNMT1 (1:1000, rabbit), DNMT3a (1:1000, rabbit) (nos. 8768, 5032 and 3598; Cell Signaling Technology, Tokyo, Japan), DNMT3b (1 μg/ml, rabbit), ERRγ (1:1000, rabbit) and β-actin (1:10,000, mouse) (ab16049, ab128930 and ab6276; Abcam, Cambridge, MA, USA).
7
Western Blot Analysis of Proteins in HCC
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Proteins from HCC cells and tissues were extracted with RIPA lysis buffer, resolved by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and transferred to a polyvinylidene fluoride membrane (Millipore, USA). The membranes were blocked with 5% non-fat powdered milk at room temperature for 1 h. The membranes were probed at 4 °C overnight with the following specific primary antibodies: DNMT3B (1:1000, Abcam, USA), LATS1 (1:5000, Abcam, USA), p-YAP (1:5000, Abcam, USA), YAP (1:5000, Abcam, USA), BAX (1:5000, Abcam, USA), BCL-2 (1:1000, Abcam, USA), KI67 (1:1000, Abcam, USA), and β-actin (1:5000, Abcam, USA). The membranes were then incubated with the appropriate secondary antibodies. Protein expression levels were visualized using an enhanced chemiluminescence detection system.
8
Protein Expression Analysis by Western Blot
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The Soluble protein components of the cells were collected after cell lysis. The protein concentration was determined by the Bradford assay (Bio-Rad). Equal amounts of lysate were applied to a 10% NuPAGE Bis-Tris Gel (Invitrogen) and gel electrophoresis was performed. The lysates were then transferred onto PVDF membranes for 90mins, and then the reactive sites were blocked with the help of a 5% skimmed milk solution. The membranes were washed once with TBST, then incubated for overnight at 4°C with relevant primary antibodies (ACADS, 16623-1-AP, proteintech; DNMT1, 24206-1-AP, proteintech; DNMT3A, 19366-1-AP, proteintach; DNMT3B, ab2851, abcam). The membranes were then washed again for three times with TBST after which the secondary antibody (Goat Anti-Rabbit IgG 1:5000) was added and the samples were incubated for a duration of 2 hours at room temperature. Upon the conclusion of this step, the membranes were subjected to three washes with TBST and then suitable amounts of ECL liquid was added and the membranes were placed in a darkroom for the reaction to proceed. A GAPDH solution (1:5000 dilution, Sigma-Aldrich) was used as the loading control reagent.
9
Quantifying DNMT Protein Levels
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10
ChIP-seq analysis of AML1, ETO, and epigenetic regulators
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Formaldehyde (1% final concentration) was added into cells (2 × 106 cells). Cells were then incubated for 10 min at 37°C to crosslink proteins to DNA. After sonication, 5 μg of antibodies recognizing the following AML1 (Santa Cruz Biotechnology, Santa Cruz, USA, sc-28679), ETO (Santa Cruz Biotechnology, Santa Cruz, USA, sc-9737), histone deacetylase 1 (HDAC1) (Abcam, Cambridge, USA, ab7028), DNA methyltransferase 1 (DNMT1) (Abcam, Cambridge, USA, ab13537), DNMT3a (Abcam, Cambridge, USA, ab13888) and DNMT3b (Abcam, Cambridge, USA, ab13604) were immunoprecipitated with the chromatin overnight. Chromatin immunoprecipitation (ChIP) was performed on the naked and sonicated DNA extracted from SKNO-1, SKNO-1-siA/E, U937, and U937-A/E cell lines and then assayed with the EZ-ChIP kit (Millipore, Billerica, MA, USA, 17-371) according to the instructions of the manufacturer. Genomic EYA4 upstream regions, which were close to the putative AML1-binding site, were amplified. Primers sequences are shown in Supplementary Table 1 . GAPDH was used as a control for nonspecific precipitated sequences.
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