Dnmt3b

Manufactured by Santa Cruz Biotechnology

Sourced in United States, China

DNMT3b is a DNA methyltransferase enzyme that catalyzes the addition of methyl groups to cytosine residues in DNA. It plays a key role in the establishment and maintenance of DNA methylation patterns during development and cellular differentiation.

Automatically generated - may contain errors

Lab products found in correlation

Dnmt3a, by Santa Cruz Biotechnology (17 mentions) Dnmt1, by Santa Cruz Biotechnology (14 mentions) Pvdf membrane, by Merck Group (5 mentions) Gapdh, by Santa Cruz Biotechnology (4 mentions) Nitrocellulose membrane, by Bio-Rad (4 mentions) β actin, by Santa Cruz Biotechnology (3 mentions) Dnmt3a, by Cell Signaling Technology (3 mentions) Ripa buffer, by Cell Signaling Technology (3 mentions) Enhanced chemiluminescence kit, by Beyotime (2 mentions) Hsc70, by Santa Cruz Biotechnology (2 mentions) Dnmt3a, by Abcam (2 mentions) Dnmt1, by Cell Signaling Technology (2 mentions) Allprep dna rna protein midi kit, by Qiagen (2 mentions) Multi gauge software, by Fujifilm (2 mentions) Las 3000 gel imager, by Fujifilm (2 mentions) Actin, by Santa Cruz Biotechnology (2 mentions) Gapdh, by Novus Biologicals (2 mentions) Brca1, by Santa Cruz Biotechnology (2 mentions) Lsm 710, by Zeiss (2 mentions) Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)

34 protocols using dnmt3b

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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Modulating DNMT3b Expression in Cells

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For transient transfection with miRs, cells at 50% confluence were transfected using Oligofectamine (Life Technologies Milan Italy) with 100 nM of pre-miR-221, scrambled, or anti miR-221 (Ambion, Life Technologies Milan Italy). In order to overexpress DNMT3b, cells were transfected with Lipofectamine 2000 and 3 μg of DNMT3b cDNA, a kind gift of Ana Portela (IDIBELL, Barcelona, Spain). To transiently knockdown DNMT3b gene expression, a pool of DNMT3b siRNAs is transfected using Lipopfectamine 2000 at a final concentration of 100 nM (Santa Cruz Biotechnology, MA, USA). To stably knockdown DNMT3b, cells were infected with a shDNMT3b (Santa Cruz Biotechnology, MA, USA) and the expression of DNMT3b studied by qRT-PCR in pooled cell populations (data not shown).

Roscigno G., Quintavalle C., Donnarumma E., Puoti I., Diaz-Lagares A., Iaboni M., Fiore D., Russo V., Todaro M., Romano G., Thomas R., Cortino G., Gaggianesi M., Esteller M., Croce C.M, & Condorelli G. (2015). MiR-221 promotes stemness of breast cancer cells by targeting DNMT3b. Oncotarget, 7(1), 580-592.

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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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Western Blot Analysis of Protein Targets

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Western blots were performed as previously described [21] (link), using the following antibodies detecting: Akt (cat.# 4691, Cell signalling, dilution 1:1000), Dnmt1 (cat.# ab188453, Abcam; dilution 1:500), Dnmt3a (cat.# SC-20703, Santa Cruz; dilution 1:1000), Dnmt3b (cat.# PA1–884, Thermo Fisher; dilution 1:1000), Stat3 (cat.# SC-8019, Santa Cruz; dilution 1:500), P-Stat3 (cat.# 9145, Cell signalling, dilution 1:1000), Stat5 (cat.# 25656, Cell signalling, dilution 1:1000), P-Stat5 (cat.# 9359, Cell signalling, dilution 1:1000), mouse c-Met (cat.# SC-8057; Santa Cruz; dilution 1:500), c-Myc (cat.# SC-40, Santa Cruz; dilution 1:1000), Hsc-70 (cat.# SC-7298, Santa Cruz; dilution 1:10000), Lin28b (cat.# SC-293120, Santa Cruz; dilution 1:500), h-Ras (cat.# SC-520, Santa Cruz, dilution 1:1000) and β-actin (cat.# SC-130657, Santa Cruz; dilution 1:1000).

Lopusna K., Nowialis P., Opavska J., Abraham A., Riva A, & Opavsky R. (2021). Dnmt3b catalytic activity is critical for its tumour suppressor function in lymphomagenesis and is associated with c-Met oncogenic signalling. EBioMedicine, 63, 103191.

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Protein Isolation and Western Blot Analysis

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Total protein was isolated using the AllPrep DNA/RNA/Protein Midi Kit
(Qiagen, Valencia, CA) according to manufacturer’s instructions. For
Western blot analysis, 20–40 μg of total or nuclear protein was
loaded on a 12% acrylamide gel, transferred to a polyvinylidene difluoride
membrane, and then blocked in 5% non-fat milk. Primary antibodies against DNMT3A
(sc-373905), DNMT3B (sc-376043), MYB (05175 MI), HbF (sc-21756), and HbA
(sc-21757) purchased from Santa Cruz Biotechnology (Dallas, TX) were diluted in
the range of 1:250 to 1:2000, incubated overnight and then followed by treatment
with secondary antibody. The immunoblots were developed using
SuperSignal® West Pico Chemiluminescent Substrate (Thermo Scientific) and
analysed on a LAS-3000 gel imager (Fujifilm Medical Systems, Stamford, CT).
Blots were processed with Restore™ Plus Western blot stripping buffer and
probed with β-actin (also termed ACTB, sc-53646) antibody as the internal
control. The band intensity of different proteins was quantified by densitometry
with MultiGauge Software (Fujifilm Medical Systems) and normalized to the band
intensity of β-actin.

Starlard‐Davenport A., Smith A., Vu L., Li B, & Pace B.S. (2019). MIR29B mediates epigenetic mechanisms of HBG gene activation. British Journal of Haematology, 186(1), 91-100.

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GR Immunoprecipitation and Immunoblotting

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Total protein was isolated in lysis buffer as previously described (14 (link)). Lysate was precleaned with protein A magnetic beads (Cell Signaling Technology) at 4°C for 1 h. GC receptor (GR) primary antibody (2 μg) (Cell Signaling Technology) was added to lysate containing 500 μg total proteins, and the mixture was gently rotated overnight at 4°C. After that, precleaned protein A magnetic beads were mixed into lysate and rotated for 1 h at room temperature. Samples were followed with 3× cold PBS washing, and proteins on beads were eluted for immunoblotting analyses of GR, DNMT3b (Santa Cruz Biotechnology), DNMT3a (Cell Signaling Technology) and DNMT1 (Cell Signaling Technology).

Chen Y.T., Hu Y., Yang Q.Y., Son J.S., Liu X.D., de Avila J.M., Zhu M.J, & Du M. (2020). Excessive Glucocorticoids During Pregnancy Impair Fetal Brown Fat Development and Predispose Offspring to Metabolic Dysfunctions. Diabetes, 69(8), 1662-1674.

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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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Immunoblotting for Protein Detection in Fat Tissues

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Immunoblotting for protein detection was conducted as we described [27 (link),32 (link)]. Fat tissues were homogenized in a modified radioimmunoprecipitation assay (RIPA) lysis buffer supplemented with 1% protease inhibitor mixture and 1% phosphatase inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA). Tissue lysates were resolved by SDS-PAGE gels. Proteins on the gels were transferred to nitrocellulose membranes (Bio-Rad, Hercules, CA, USA), which were then blocked, washed, and incubated with various primary antibodies, followed by Alexa Fluor 680-conjugated secondary antibodies (ThermoFisher Scientific). The blots were developed with a Li-COR Imager System (Li-COR Biosciences, Lincoln, NE, USA). Primary antibodies used were as follows: Anti-UCP1 antibody (1:500, ab23841, Abcam, Cambridge, MA, USA); Anti-α-Tubulin antibody (1:1000, ABCENT4777, Advanced BioChemicals, Lawrenceville, GA, USA); Mitochondrial total OXPHOS protein antibody set (Abcam,ab110413); and Anti-pHSL (4126s, Cell Signaling Technology, Danvers, MA, USA); DNMT3b (sc-393845, Santa Cruz, Dallas, TX, USA, sc-393845); HSL (Cell Signaling, 4107s).

Wang S., Cao Q., Cui X., Jing J., Li F., Shi H., Xue B, & Shi H. (2021). Dnmt3b Deficiency in Myf5+-Brown Fat Precursor Cells Promotes Obesity in Female Mice. Biomolecules, 11(8), 1087.

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Western Blot Analysis of Protein Markers

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The 30-µg protein extracts were mixed with a sample buffer, boiled for 10 min and followed by electrophoresis using 8%–15% sodium dodecyl sulfate-polyacrylamide gels. The proteins in the gels were transferred to a polyvinylidene difluoride membrane. Blots were incubated with primary antibodies against collagen-1α1 (Santa Cruz), DNMT1 (Santa Cruz), DNMT3b (Santa Cruz), SET1A (Santa Cruz), PTEN (Santa Cruz), PI3K (PROTEINTECH, Rosemont, IL, USA) and GAPDH (Santa Cruz) for protein control. After washing the blots with tris-buffered saline and incubating them with horseradish peroxidase-coupled anti-rabbit immunoglobulin-G antibodies (dilution, 1:5000), HRP anti-mouse immunoglobulin-G antibodies (dilution, 1:10,000) and HRP anti-goat immunoglobulin-G antibodies (dilution, 1:10,000) at room temperature for 1 h, we developed them with enhanced chemiluminescence detection (GE Healthcare Biosciences AB, Uppsala, Sweden), exposed them to film and quantified the signals using densitometry.

Yang Y.L., Wang F.S., Li S.C., Tiao M.M, & Huang Y.H. (2017). MicroRNA-29a Alleviates Bile Duct Ligation Exacerbation of Hepatic Fibrosis in Mice through Epigenetic Control of Methyltransferases. International Journal of Molecular Sciences, 18(1), 192.

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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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