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Dnmt3a

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DNMT3a is a DNA methyltransferase enzyme that catalyzes the addition of methyl groups to cytosine residues in DNA. It is involved in the establishment of DNA methylation patterns during development and cellular differentiation.

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Lab products found in correlation

Dnmt3b, by Santa Cruz Biotechnology (17 mentions) Dnmt1, by Santa Cruz Biotechnology (16 mentions) β actin, by Santa Cruz Biotechnology (7 mentions) Pvdf membrane, by Merck Group (4 mentions) Gapdh, by Santa Cruz Biotechnology (3 mentions) Ripa buffer, by Cell Signaling Technology (3 mentions) Enhanced chemiluminescence kit, by Beyotime (2 mentions) Vimentin, by Cell Signaling Technology (2 mentions) Tubulin, by Merck Group (2 mentions) 2100 antigen retriever in buffer a, by Electron Microscopy Sciences (2 mentions) Caspase 8, by Cell Signaling Technology (2 mentions) Caspase 3, by Cell Signaling Technology (2 mentions) β actin, by Merck Group (2 mentions) Hdac2, by Santa Cruz Biotechnology (2 mentions) Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (2 mentions) Gapdh, by Novus Biologicals (2 mentions) Brca1, by Santa Cruz Biotechnology (2 mentions) Protease inhibitor cocktail, by Roche (2 mentions) E cadherin, by BD (2 mentions) Nitrocellulose membrane, by Bio-Rad (2 mentions)

38 protocols using dnmt3a

1

Epigenetic Profiling of DES Treatment

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DES was purchased from Santa Cruz Biotechnology, Inc. (CA, USA), diluted in DMSO(dimethylsulfoxide) to 500 M and stored at -20°C. The final concentrations used here were 2×10−7, 2×10−6, and 2×10−5 M, and they were freshly diluted with DMEM to their final concentrations. Controls were treated with the same amount of DMSO (0.04%) used in the corresponding experiment. A TRIzol Reagent Kit was obtained from Invitrogen (Carlsbad, CA, USA), and a PrimeScript RT Reagent Kit was purchased from Takara (Otsu, Japan). GoTaq Hot Start Green Master Mix was obtained from Promega (Wisconsin, USA). Dnmt1, Dnmt3a, and Dnmt3b antibodies were obtained from Santa Cruz Biotechnology, Inc. An HRP-conjugated(horseradish peroxidase-conjugated) secondary antibody, an enhanced chemiluminescence kit and an Annexin V–FITC(fluorescein isothiocyanate) Apoptosis Detection Kit were purchased from Beyotime (Shanghai, China). An EDU(5-Ethynyl -2’- deoxyuridine) Cell Proliferation Kit was obtained from Ribo (Guangzhou, China), and an EZ DNA Methylation-Gold Kit was purchased from Zymo Research (Orange, CA, USA).
Yin L., Zheng L.J., Jiang X., Liu W.B., Han F., Cao J, & Liu J.Y. (2015). Effects of Low-Dose Diethylstilbestrol Exposure on DNA Methylation in Mouse Spermatocytes. PLoS ONE, 10(11), e0143143.
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2

Immunoblot Analysis of Key Proteins

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Immunoblot analysis was done as previously described (19 (link)) using antibodies directed against ATDC (Santa Cruz Biotechnology), PTEN and RELA (Cell Signaling Technology, Danvers, MA), DNMT3A and DNMT3B (Santa Cruz Biotechnology), at a 1:1000 dilution. β-actin antibody (Sigma, St Louis, MD) served as a loading control.
Palmbos P.L., Wang L., Yang H., Wang Y., Leflein J., Ahmet M.L., Wilkinson J.E., Kumar-Sinha C., Ney G.M., Tomlins S.A., Daignault S., Kunju L.P., Wu X.R., Lotan Y., Liebert M., Ljungman M.E, & Simeone D.M. (2015). ATDC/TRIM29 drives invasive bladder cancer formation through microRNA-mediated and epigenetic mechanisms. Cancer research, 75(23), 5155-5166.
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3

Tamoxifen and 5-aza Modulate Epigenetic Markers

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Tamoxifen (4-hydroxyTamoxifen, 4-OHT/TAM) and 5-aza (5-aza-2′-deoxycytidine, 5-aza-dC) were purchased from Sigma-Aldrich (St Louis, Missouri, USA). Indicated cells were treated with 5 μmol/L TAM for 48 h. E-cadherin, vimentin, ER-α, PR, and Her-2 antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Actin, p-gp, DNMT1 and DNMT3a antibodies were purchased from Santa Cruz Biotechnology (USA).
Wang Q., Cheng Y., Wang Y., Fan Y., Li C., Zhang Y., Wang Y., Dong Q., Ma Y., Teng Y.E., Qu X, & Liu Y. (2017). Tamoxifen reverses epithelial–mesenchymal transition by demethylating miR-200c in triple-negative breast cancer cells. BMC Cancer, 17, 492.
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4

Immunoblot Analysis of DNMT3A and Tubulin

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The protocol was used as previously described [4 (link)]. All primary antibodies were incubated with the membrane at 4°C overnight: DNMT3A (Santa Cruz Biotechnology, sc-20703), and tubulin (Sigma, T6199). Membranes were washed with 1×TBST and incubated with either anti-mouse or anti-rabbit IgG horseradish peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology).
Zhu Y., Zheng G., Wang H., Jia Y., Zhang Y., Tang Y., Li W., Fan Y., Zhang X., Liu Y, & Liu S. (2017). Downregulated miR-29a/b/c during Contact Inhibition Stage Promote 3T3-L1 Adipogenesis by Targeting DNMT3A. PLoS ONE, 12(1), e0170636.
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5

Western Blot Analysis of Cellular Proteins

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After the various treatments, the cells were collected into 1 × cell lysis buffer41 (link) and the western blotting was performed with whole-cell lysates as previously described.41 (link) Equivalent gel loading was confirmed by probing with β-actin antibody. The antibodies used were: β-actin (1 : 1000) and DNMT3a (1 : 1000) (Santa Cruz Biotechnology); EZH2 (1 : 500) and cleaved caspase-3 (1 : 500) (Cell Signaling Technology, Danvers, MA, USA).
Yan F., Shen N., Pang J., Xie D., Deng B., Molina J.R., Yang P, & Liu S. (2014). Restoration of miR-101 suppresses lung tumorigenesis through inhibition of DNMT3a-dependent DNA methylation. Cell Death & Disease, 5(9), e1413-.
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6

Oxidative Stress and Epigenetic Regulators

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Cells were washed with ice-cold PBS and lysed on ice with a protease inhibitor cocktail. Protein concentrations were measured by BCA method. Protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes. The membranes were probed with NRF2 (1:500), HO1 (1:1000), SOD1 (1:1000), GST (1:1000), DNMT1 (1:500), DNMT3a (1:500), DNMT3b (1:500), MeCP2 (1:500), MBD2 (1:500), GAPDH (1:10000) antibodies (Santa Cruz Biotechnology, Inc., Texas, United States) at 4°C overnight. The bands were visualized after incubation with a chemiluminescent substrate. Quantification of the band density was determined by densitometric analysis.
Lou D., Wei X., Xiao P., Huo Q., Hong X., Sun J., Shuai Y, & Tao G. (2020). Demethylation of the NRF2 Promoter Protects Against Carcinogenesis Induced by Nano-SiO2. Frontiers in Genetics, 11, 818.
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7

Comprehensive Immunohistochemical Analysis of Intestinal Tissue

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Tissues were isolated and fixed using 4% paraformaldehyde and then embedded in paraffin. Antigen retrieval was performed using the 2100 Antigen Retriever in buffer A (Electron Microscopy Sciences), and standard immunostaining procedures were performed for Dnmt1 (Santa Cruz Biotechnology), Chga (Immunostar), Dnmt3a (Santa Cruz Biotechnology), Dnmt3b (Abcam), E-Cadherin (BD Transduction Laboratories), Sox9 (Millipore), Lyz (Dako), Ki67 (BD Pharmingen), and Muc2 (Santa Cruz Biotechnology). Immunohistochemical procedures were modified for Hes-1 (Ben Stanger, University of Pennsylvania), including antigen retrieval in high-pH antigen unmasking solution (Vector Laboratories) and signal amplification with the TSA Fluorescein system (Perkin Elmer). AP staining was performed using NBT and BCIP (Boehringer Ingelheim). In situ hybridization for Olfm4 was performed as described previously (Barker et al. 2007 (link)). All microscopy was performed on the Nikon Eclipse 80i.
Sheaffer K.L., Kim R., Aoki R., Elliott E.N., Schug J., Burger L., Schübeler D, & Kaestner K.H. (2014). DNA methylation is required for the control of stem cell differentiation in the small intestine. Genes & Development, 28(6), 652-664.
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8

Adipogenesis Protein Expression Analysis

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Cell and tissue protein was extracted and Western Blotting performed as described previously. 36 For adipose tissue, protein expression analysis included preadipocyte marker (Pref-1, 55 kDa, Millipore); suppressor of preadipocyte differentiation (SOX9, Millipore); adipogeneic markers (PPARγ, 57 kDa, Cayman; C/EBPα, 42 kDa, Santa Cruz); lipogenic factor (SREBP1, 125 kDa, Santa Cruz), and; epigenetic regulators DNA methyltransferase 3a (DNMT3a, 120 kDa, Santa Cruz) and lysine (K)-specific demethylase 1A (LSD1, 110 kDa, Cell Signaling).
Desai M., Ferrini M.G., Jellyman J.K., Han G, & Ross M.G. (2018). In Vivo and In Vitro Bisphenol A Exposure Effects on Adiposity. Journal of developmental origins of health and disease, 9(6), 678-687.
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9

Proteasome Inhibition Modulates Epigenetic Regulators

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HLECs were precultured with 2.5 μM and 5 μM MG-132 (Selleck Chemicals, Houston, TX), a proteasome protease inhibitor for 4 h, then, followed by a washout in regular medium, with or without 100 μM MGO for 24 h. The harvested cells were lysed using RIPA buffer (Cell Signaling Technology, Inc. Beverly, MA) and the proteins were analyzed by Western blotting using the antibodies specific to Nrf2, Dnmt1, Dnmt3a and Dnmt3b (Santa Cruz Biotechnology, Santa Cruz, CA).
Palsamy P., Bidasee K.R., Ayaki M., Augusteyn R.C., Chan J.Y, & Shinohara T. (2014). Methylglyoxal induces endoplasmic reticulum stress and DNA demethylation in the Keap1 promoter of human lens epithelial cells and age-related cataracts. Free radical biology & medicine, 72, 134-148.
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10

Western Blotting of DNA Methyltransferases

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Western blots were performed as previously described (31 (link)), using the following antibodies: Dnmt1 (sc271729, Santa Cruz; dilution 1:1000), Dnmt3a (sc-20703, Santa Cruz; dilution 1:1000), Dnmt3b (PA1-884, Thermo Fisher; dilution 1:1000), p53 (sc6243, Santa Cruz; dilution 1:1000), Hsc70 (sc-7298, Santa Cruz; dilution 1:10,000). Uncropped and unprocessed scans of blots are available in Source Data file.
Lopusna K., Nowialis P., Opavska J., Abraham A., Riva A., Haney S.L, & Opavsky R. (2021). Decreases in different Dnmt3b activities drive distinct development of hematologic malignancies in mice. The Journal of Biological Chemistry, 296, 100285.
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