primary antibodies against dnmt3a Search Results


gene exp dnmt3a hs00173377 m1  (Thermo Fisher)
86
Thermo Fisher gene exp dnmt3a hs00173377 m1
Gene Exp Dnmt3a Hs00173377 M1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse monoclonal antibodies against dnmt3a  (Novus Biologicals)
94
Novus Biologicals mouse monoclonal antibodies against dnmt3a
Fig. 2. Immunological detection of <t>DNMT3A</t> protein in seminoma (SE), non-seminoma (NS) and adja
Mouse Monoclonal Antibodies Against Dnmt3a, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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the catalytic domain of human dnmt3a (cd-dnmt3a; amino acids 611–912)  (Millipore)
90
Millipore the catalytic domain of human dnmt3a (cd-dnmt3a; amino acids 611–912)
Fig. 2. Immunological detection of <t>DNMT3A</t> protein in seminoma (SE), non-seminoma (NS) and adja
The Catalytic Domain Of Human Dnmt3a (Cd Dnmt3a; Amino Acids 611–912), supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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aav-spguide  (Nature Biotechnology)
90
Nature Biotechnology aav-spguide
Fig. 2. Immunological detection of <t>DNMT3A</t> protein in seminoma (SE), non-seminoma (NS) and adja
Aav Spguide, supplied by Nature Biotechnology, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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mouse monoclonal anti dnmt3a  (Cell Signaling Technology Inc)
96
Cell Signaling Technology Inc mouse monoclonal anti dnmt3a
<t>Dnmt3a</t> is present in skeletal muscle and CNS mitochondria of adult mouse and human . (A) Western blots showing that Dnmt3a, but not Dnmt1, is present in adult mouse skeletal muscle mitochondria. Dnmt1 was present in crude tissue fractions but was undetectable in pure mitochondrial fractions even with prolonged overexposures as shown. Western blot for the mitochondrial marker complex V is used to verify mitochondrial enrichment of the fraction. Ponceau S-stained membrane is used to show protein loading. (B) Experiments that establish that Dnmt1 is bound to the outer surface of mitochondria. Freshly isolated intact skeletal muscle mitochondria were treated (+) with agarose-conjugated proteinase K to digest surface-bound proteins or were not treated with proteinase K (−). Mitochondria were lysed and proteins were fractionated by SDS-PAGE and western blotted for Dnmt1 using two different antibodies that detect different amino acid (aa) domains of the protein. Protein loading was revealed by reprobing the blots for complex V. Crude nuclear fractions were used as a positive control. (C) Experiments that establish that Dnmt3a is within mitochondria and not merely bound to the outer surface of mitochondria. Freshly isolated intact skeletal muscle mitochondria were treated with agarose-conjugated proteinase K to digest surface-bound proteins. The efficacy of digestion is shown by the loss of desmin and tubulin/MAP immunoreactivities in the treated samples, but Dnmt3a immunoreactivity was not attenuated by proteinase K digestion. Protein loading is shown by Ponceau S staining of membranes. Mitochondrial enrichment is shown by complex V immunoreactivity. (D) Adult mouse brain and spinal cord were homogenized, fractionated and immunoblotted with antibody to Dnmt3a. Lanes were loaded with equivalent amounts of protein from the crude homogenate (H) and different fractions, including nuclear-enriched and insoluble material (P1), post-nuclear supernatant (S1), crude mitochondria (P2), post-mitochondrial supernatant (S2), and pure mitochondria (PM). See Figure for the validation of this fractionation method. The 100 kDa form of Dnmt3a was present in all fractions, including the pure mitochondria, while the 78 kDa form was present in all fractions except the mitochondria. (E) Western blot comparing the levels of Dnmt3a and isoform specificity in skeletal muscle (100 μg protein) and spinal cord (50 μg protein). Dnmt3a expression is greater in spinal cord compared to skeletal muscle. The 78 kDa isoform predominates in skeletal muscle while the 100 kDa isoform predominates in spinal cord. (F) Graph showing the relative levels of Dnmt3a isoform immunoreactivity determined by immunobloting of pure mitochondria isolated from adult mouse skeletal muscle and spinal cord. Values are mean ± SD. The 100 kDa isoform was not detected in skeletal muscle even after long exposures. (G) Immunoblot for Dnmt3a in pure mitochondria (PM) fractions from different types of adult mouse tissues, human brain, and a human cell line. The 100 kDa isoform of Dnmt3a was present in mouse spinal cord, brain, heart, testes, and human brain cerebral cortex. Dnmt3a immunoreactivity was low or undetectable in mouse spleen, liver, kidney, and lung and in human embryonic kidney cells. Western blot for the mitochondrial marker cyclophilin D is used to show protein loading.
Mouse Monoclonal Anti Dnmt3a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 96/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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gene exp dnmt3a rn01027162 g1  (Thermo Fisher)
85
Thermo Fisher gene exp dnmt3a rn01027162 g1
Correlation analysis between plasma homocysteine levels (μmol/L) and hepatic <t>Dnmt3a</t> mRNA levels (AU). Male rats are represented by squares and females by crosses.
Gene Exp Dnmt3a Rn01027162 G1, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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snp dnmt3a c 7863728 10  (Thermo Fisher)
85
Thermo Fisher snp dnmt3a c 7863728 10
Association analysis between polymorphisms of <t> DNA methyltransferase </t> genes and risk of grade ≥ 2 radiation-induced fibrosis (LENT-SOMA scale) in breast cancer patients
Snp Dnmt3a C 7863728 10, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 85/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dnmt3a antibody  (Cell Signaling Technology Inc)
90
Cell Signaling Technology Inc dnmt3a antibody
(A) MSP showed that the methylation level in the pre-miR-145 promoter of SKOV3 and 3AO cells was decreased by 20(S)-Rg3. M: methylated product, U: unmethylated product. Fisher exact test. (B) Western blot analysis indicated that <t>DNMT3A</t> rather than DNMT3B and DNMT1 was significantly decreased in SKOV3 and 3AO cells treated by 20(S)-Rg3. (C) qRT-PCR showed that transfection of DNMT3A plasmid rescued DNMT3A level in 20(S)-Rg3-treated cells. t test. (D) qRT-PCR showed overexpression of DNMT3A reversed 20(S)-Rg3-induced miR-145. (E) MSP showed the methylation level in the pre-miR-145 promoter was increased in cells treated with both 20(S)-Rg3 and DNMT3A plasmid compared to that in cells treated with 20(S)-Rg3 alone. M: methylated product, U: unmethylated product. Fisher exact test. (F) Western blot analysis showed that restoration of DNMT3A reversed 20(S)-Rg3-induced E-cadherin increase, N-cadherin and vimentin decrease. (G) In vitro migration assay showed that transfection of DNMT3A plasmid stimulated motility and invasion of 20(S)-Rg3-treated cells (200×). t test. All experiments were performed in triplicate and data were showed as means ± SE.
Dnmt3a Antibody, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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anti dnmt3a  (Novus Biologicals)
92
Novus Biologicals anti dnmt3a
(A) MSP showed that the methylation level in the pre-miR-145 promoter of SKOV3 and 3AO cells was decreased by 20(S)-Rg3. M: methylated product, U: unmethylated product. Fisher exact test. (B) Western blot analysis indicated that <t>DNMT3A</t> rather than DNMT3B and DNMT1 was significantly decreased in SKOV3 and 3AO cells treated by 20(S)-Rg3. (C) qRT-PCR showed that transfection of DNMT3A plasmid rescued DNMT3A level in 20(S)-Rg3-treated cells. t test. (D) qRT-PCR showed overexpression of DNMT3A reversed 20(S)-Rg3-induced miR-145. (E) MSP showed the methylation level in the pre-miR-145 promoter was increased in cells treated with both 20(S)-Rg3 and DNMT3A plasmid compared to that in cells treated with 20(S)-Rg3 alone. M: methylated product, U: unmethylated product. Fisher exact test. (F) Western blot analysis showed that restoration of DNMT3A reversed 20(S)-Rg3-induced E-cadherin increase, N-cadherin and vimentin decrease. (G) In vitro migration assay showed that transfection of DNMT3A plasmid stimulated motility and invasion of 20(S)-Rg3-treated cells (200×). t test. All experiments were performed in triplicate and data were showed as means ± SE.
Anti Dnmt3a, supplied by Novus Biologicals, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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epiquick dnmt1 and dnmt3a assay kit  (EpiGentek)
90
EpiGentek epiquick dnmt1 and dnmt3a assay kit
(A) MSP showed that the methylation level in the pre-miR-145 promoter of SKOV3 and 3AO cells was decreased by 20(S)-Rg3. M: methylated product, U: unmethylated product. Fisher exact test. (B) Western blot analysis indicated that <t>DNMT3A</t> rather than DNMT3B and DNMT1 was significantly decreased in SKOV3 and 3AO cells treated by 20(S)-Rg3. (C) qRT-PCR showed that transfection of DNMT3A plasmid rescued DNMT3A level in 20(S)-Rg3-treated cells. t test. (D) qRT-PCR showed overexpression of DNMT3A reversed 20(S)-Rg3-induced miR-145. (E) MSP showed the methylation level in the pre-miR-145 promoter was increased in cells treated with both 20(S)-Rg3 and DNMT3A plasmid compared to that in cells treated with 20(S)-Rg3 alone. M: methylated product, U: unmethylated product. Fisher exact test. (F) Western blot analysis showed that restoration of DNMT3A reversed 20(S)-Rg3-induced E-cadherin increase, N-cadherin and vimentin decrease. (G) In vitro migration assay showed that transfection of DNMT3A plasmid stimulated motility and invasion of 20(S)-Rg3-treated cells (200×). t test. All experiments were performed in triplicate and data were showed as means ± SE.
Epiquick Dnmt1 And Dnmt3a Assay Kit, supplied by EpiGentek, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dnmt3a  (Cell Signaling Technology Inc)
92
Cell Signaling Technology Inc dnmt3a
(A) MSP showed that the methylation level in the pre-miR-145 promoter of SKOV3 and 3AO cells was decreased by 20(S)-Rg3. M: methylated product, U: unmethylated product. Fisher exact test. (B) Western blot analysis indicated that <t>DNMT3A</t> rather than DNMT3B and DNMT1 was significantly decreased in SKOV3 and 3AO cells treated by 20(S)-Rg3. (C) qRT-PCR showed that transfection of DNMT3A plasmid rescued DNMT3A level in 20(S)-Rg3-treated cells. t test. (D) qRT-PCR showed overexpression of DNMT3A reversed 20(S)-Rg3-induced miR-145. (E) MSP showed the methylation level in the pre-miR-145 promoter was increased in cells treated with both 20(S)-Rg3 and DNMT3A plasmid compared to that in cells treated with 20(S)-Rg3 alone. M: methylated product, U: unmethylated product. Fisher exact test. (F) Western blot analysis showed that restoration of DNMT3A reversed 20(S)-Rg3-induced E-cadherin increase, N-cadherin and vimentin decrease. (G) In vitro migration assay showed that transfection of DNMT3A plasmid stimulated motility and invasion of 20(S)-Rg3-treated cells (200×). t test. All experiments were performed in triplicate and data were showed as means ± SE.
Dnmt3a, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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dna methyltransferase genes  (Thermo Fisher)
99
Thermo Fisher dna methyltransferase genes
(A) MSP showed that the methylation level in the pre-miR-145 promoter of SKOV3 and 3AO cells was decreased by 20(S)-Rg3. M: methylated product, U: unmethylated product. Fisher exact test. (B) Western blot analysis indicated that <t>DNMT3A</t> rather than DNMT3B and DNMT1 was significantly decreased in SKOV3 and 3AO cells treated by 20(S)-Rg3. (C) qRT-PCR showed that transfection of DNMT3A plasmid rescued DNMT3A level in 20(S)-Rg3-treated cells. t test. (D) qRT-PCR showed overexpression of DNMT3A reversed 20(S)-Rg3-induced miR-145. (E) MSP showed the methylation level in the pre-miR-145 promoter was increased in cells treated with both 20(S)-Rg3 and DNMT3A plasmid compared to that in cells treated with 20(S)-Rg3 alone. M: methylated product, U: unmethylated product. Fisher exact test. (F) Western blot analysis showed that restoration of DNMT3A reversed 20(S)-Rg3-induced E-cadherin increase, N-cadherin and vimentin decrease. (G) In vitro migration assay showed that transfection of DNMT3A plasmid stimulated motility and invasion of 20(S)-Rg3-treated cells (200×). t test. All experiments were performed in triplicate and data were showed as means ± SE.
Dna Methyltransferase Genes, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Image Search Results


Fig. 2. Immunological detection of DNMT3A protein in seminoma (SE), non-seminoma (NS) and adja

Journal: The Tohoku journal of experimental medicine

Article Title: Up-regulation of DNA-methyltransferase 3A expression is associated with hypomethylation of intron 25 in human testicular germ cell tumors.

doi: 10.1620/tjem.212.177

Figure Lengend Snippet: Fig. 2. Immunological detection of DNMT3A protein in seminoma (SE), non-seminoma (NS) and adja

Article Snippet: The primary antibodies used were 200- and 400-fold dilutions of mouse monoclonal antibodies against DNMT3A (64B1446, Imgenex Co., San Diego, CA, USA) and γ - tubulin (GTU-88, Sigma-Aldrich, St. Louis, MO, USA), respectively.

Techniques:

Fig. 3. Analyses of DNMT3A methylation status. A: Methylation status of CpGs in the sequences

Journal: The Tohoku journal of experimental medicine

Article Title: Up-regulation of DNA-methyltransferase 3A expression is associated with hypomethylation of intron 25 in human testicular germ cell tumors.

doi: 10.1620/tjem.212.177

Figure Lengend Snippet: Fig. 3. Analyses of DNMT3A methylation status. A: Methylation status of CpGs in the sequences

Article Snippet: The primary antibodies used were 200- and 400-fold dilutions of mouse monoclonal antibodies against DNMT3A (64B1446, Imgenex Co., San Diego, CA, USA) and γ - tubulin (GTU-88, Sigma-Aldrich, St. Louis, MO, USA), respectively.

Techniques: Methylation

Dnmt3a is present in skeletal muscle and CNS mitochondria of adult mouse and human . (A) Western blots showing that Dnmt3a, but not Dnmt1, is present in adult mouse skeletal muscle mitochondria. Dnmt1 was present in crude tissue fractions but was undetectable in pure mitochondrial fractions even with prolonged overexposures as shown. Western blot for the mitochondrial marker complex V is used to verify mitochondrial enrichment of the fraction. Ponceau S-stained membrane is used to show protein loading. (B) Experiments that establish that Dnmt1 is bound to the outer surface of mitochondria. Freshly isolated intact skeletal muscle mitochondria were treated (+) with agarose-conjugated proteinase K to digest surface-bound proteins or were not treated with proteinase K (−). Mitochondria were lysed and proteins were fractionated by SDS-PAGE and western blotted for Dnmt1 using two different antibodies that detect different amino acid (aa) domains of the protein. Protein loading was revealed by reprobing the blots for complex V. Crude nuclear fractions were used as a positive control. (C) Experiments that establish that Dnmt3a is within mitochondria and not merely bound to the outer surface of mitochondria. Freshly isolated intact skeletal muscle mitochondria were treated with agarose-conjugated proteinase K to digest surface-bound proteins. The efficacy of digestion is shown by the loss of desmin and tubulin/MAP immunoreactivities in the treated samples, but Dnmt3a immunoreactivity was not attenuated by proteinase K digestion. Protein loading is shown by Ponceau S staining of membranes. Mitochondrial enrichment is shown by complex V immunoreactivity. (D) Adult mouse brain and spinal cord were homogenized, fractionated and immunoblotted with antibody to Dnmt3a. Lanes were loaded with equivalent amounts of protein from the crude homogenate (H) and different fractions, including nuclear-enriched and insoluble material (P1), post-nuclear supernatant (S1), crude mitochondria (P2), post-mitochondrial supernatant (S2), and pure mitochondria (PM). See Figure for the validation of this fractionation method. The 100 kDa form of Dnmt3a was present in all fractions, including the pure mitochondria, while the 78 kDa form was present in all fractions except the mitochondria. (E) Western blot comparing the levels of Dnmt3a and isoform specificity in skeletal muscle (100 μg protein) and spinal cord (50 μg protein). Dnmt3a expression is greater in spinal cord compared to skeletal muscle. The 78 kDa isoform predominates in skeletal muscle while the 100 kDa isoform predominates in spinal cord. (F) Graph showing the relative levels of Dnmt3a isoform immunoreactivity determined by immunobloting of pure mitochondria isolated from adult mouse skeletal muscle and spinal cord. Values are mean ± SD. The 100 kDa isoform was not detected in skeletal muscle even after long exposures. (G) Immunoblot for Dnmt3a in pure mitochondria (PM) fractions from different types of adult mouse tissues, human brain, and a human cell line. The 100 kDa isoform of Dnmt3a was present in mouse spinal cord, brain, heart, testes, and human brain cerebral cortex. Dnmt3a immunoreactivity was low or undetectable in mouse spleen, liver, kidney, and lung and in human embryonic kidney cells. Western blot for the mitochondrial marker cyclophilin D is used to show protein loading.

Journal: Frontiers in Cellular Neuroscience

Article Title: Mitochondrial DNMT3A and DNA methylation in skeletal muscle and CNS of transgenic mouse models of ALS

doi: 10.3389/fncel.2013.00279

Figure Lengend Snippet: Dnmt3a is present in skeletal muscle and CNS mitochondria of adult mouse and human . (A) Western blots showing that Dnmt3a, but not Dnmt1, is present in adult mouse skeletal muscle mitochondria. Dnmt1 was present in crude tissue fractions but was undetectable in pure mitochondrial fractions even with prolonged overexposures as shown. Western blot for the mitochondrial marker complex V is used to verify mitochondrial enrichment of the fraction. Ponceau S-stained membrane is used to show protein loading. (B) Experiments that establish that Dnmt1 is bound to the outer surface of mitochondria. Freshly isolated intact skeletal muscle mitochondria were treated (+) with agarose-conjugated proteinase K to digest surface-bound proteins or were not treated with proteinase K (−). Mitochondria were lysed and proteins were fractionated by SDS-PAGE and western blotted for Dnmt1 using two different antibodies that detect different amino acid (aa) domains of the protein. Protein loading was revealed by reprobing the blots for complex V. Crude nuclear fractions were used as a positive control. (C) Experiments that establish that Dnmt3a is within mitochondria and not merely bound to the outer surface of mitochondria. Freshly isolated intact skeletal muscle mitochondria were treated with agarose-conjugated proteinase K to digest surface-bound proteins. The efficacy of digestion is shown by the loss of desmin and tubulin/MAP immunoreactivities in the treated samples, but Dnmt3a immunoreactivity was not attenuated by proteinase K digestion. Protein loading is shown by Ponceau S staining of membranes. Mitochondrial enrichment is shown by complex V immunoreactivity. (D) Adult mouse brain and spinal cord were homogenized, fractionated and immunoblotted with antibody to Dnmt3a. Lanes were loaded with equivalent amounts of protein from the crude homogenate (H) and different fractions, including nuclear-enriched and insoluble material (P1), post-nuclear supernatant (S1), crude mitochondria (P2), post-mitochondrial supernatant (S2), and pure mitochondria (PM). See Figure for the validation of this fractionation method. The 100 kDa form of Dnmt3a was present in all fractions, including the pure mitochondria, while the 78 kDa form was present in all fractions except the mitochondria. (E) Western blot comparing the levels of Dnmt3a and isoform specificity in skeletal muscle (100 μg protein) and spinal cord (50 μg protein). Dnmt3a expression is greater in spinal cord compared to skeletal muscle. The 78 kDa isoform predominates in skeletal muscle while the 100 kDa isoform predominates in spinal cord. (F) Graph showing the relative levels of Dnmt3a isoform immunoreactivity determined by immunobloting of pure mitochondria isolated from adult mouse skeletal muscle and spinal cord. Values are mean ± SD. The 100 kDa isoform was not detected in skeletal muscle even after long exposures. (G) Immunoblot for Dnmt3a in pure mitochondria (PM) fractions from different types of adult mouse tissues, human brain, and a human cell line. The 100 kDa isoform of Dnmt3a was present in mouse spinal cord, brain, heart, testes, and human brain cerebral cortex. Dnmt3a immunoreactivity was low or undetectable in mouse spleen, liver, kidney, and lung and in human embryonic kidney cells. Western blot for the mitochondrial marker cyclophilin D is used to show protein loading.

Article Snippet: The membranes were stained with Ponceau S (Sigma) to determine transfer efficiency, destained, blocked with 1% BSA/0.05% Tween 20/TBS and incubated with primary antibodies: rabbit polyclonal anti-cytochrome P450 reductase (Stressgen) at 1:1000; mouse monoclonal anti-actin (Chemicon) at 1:1000; mouse monoclonal anti-porin (Mitosciences) at 1:3000; rabbit polyclonal anti-lamin (Millipore) at 1:500; sheep polyclonal anti-catalase (Biodesign International) at 1:2000; mouse anti-cyclophilin D (Mitosciences) at 1:3000; mouse monoclonal anti-complex V (Molecular Probes Invitrogen) at 1:10,000; rabbit polyclonal and mouse monoclonal anti-Dnmt3a (Cell Signaling, Abgent, and Alexis) at 1:100–1:500, rabbit polyclonal and mouse monoclonal anti-Dnmt1 (Bethyl, Novus, and Alexis) at 1:500–1:5000, and rabbit polyclonal antibody to Dnmt3b (Abcam) at 1:250.

Techniques: Western Blot, Marker, Staining, Isolation, SDS Page, Positive Control, Fractionation, Expressing

Antibodies to Dnmt isoforms screened in this study .

Journal: Frontiers in Cellular Neuroscience

Article Title: Mitochondrial DNMT3A and DNA methylation in skeletal muscle and CNS of transgenic mouse models of ALS

doi: 10.3389/fncel.2013.00279

Figure Lengend Snippet: Antibodies to Dnmt isoforms screened in this study .

Article Snippet: The membranes were stained with Ponceau S (Sigma) to determine transfer efficiency, destained, blocked with 1% BSA/0.05% Tween 20/TBS and incubated with primary antibodies: rabbit polyclonal anti-cytochrome P450 reductase (Stressgen) at 1:1000; mouse monoclonal anti-actin (Chemicon) at 1:1000; mouse monoclonal anti-porin (Mitosciences) at 1:3000; rabbit polyclonal anti-lamin (Millipore) at 1:500; sheep polyclonal anti-catalase (Biodesign International) at 1:2000; mouse anti-cyclophilin D (Mitosciences) at 1:3000; mouse monoclonal anti-complex V (Molecular Probes Invitrogen) at 1:10,000; rabbit polyclonal and mouse monoclonal anti-Dnmt3a (Cell Signaling, Abgent, and Alexis) at 1:100–1:500, rabbit polyclonal and mouse monoclonal anti-Dnmt1 (Bethyl, Novus, and Alexis) at 1:500–1:5000, and rabbit polyclonal antibody to Dnmt3b (Abcam) at 1:250.

Techniques: Recombinant, Immunohistochemistry

Dnmt3a levels are reduced in hSOD1 tg mouse models of ALS . (A) Western blot for Dnmt3a in leg skeletal muscle mitochondria (100 μg/lane) in non-conditional tg mice expressing hSOD1-G37R or hSOD1-wildtype constructs and conditional (muscle-restricted) tg mice expressing hSOD1 mus -G37R, -G93A, or -wildtype constructs compared to age-matched non-tg littermates. Ponceau S staining of membrane shows protein loading. (B) Graph showing the relative levels of Dnmt3a immunoreactivity determined by immunoblotting of pure mitochondria isolated from hSOD1 tg mouse leg skeletal muscle. Values are mean ± SD. Significant differences from non-tg control are indicated by single ( p < 0.05) or double ( p < 0.01) asterisks. (C) Western blot for Dnmt3a in spinal cord mitochondria fractions (50 μg/lane) in conditional (muscle-restricted) tg mice expressing hSOD1 mus -G37R, -G93A, or -wildtype constructs compared to age-matched non-tg littermates. Ponceau S staining of membrane shows protein loading. (D) Graph showing the relative levels of Dnmt3a immunoreactivity in hSOD1 mus tg mouse spinal cord. Values are mean ± SD. Significant differences from non-tg control are indicated by an asterisk ( p < 0.05). The results have been replicated in at least 3 different experiments. (E) Western blot for Dnmt3a in crude fractions of skeletal muscle and spinal cord of conditional (muscle-restricted) tg mice expressing hSOD1 mus -G37R or -wildtype constructs compared to age-matched non-tg littermates. Ponceau S staining of membrane shows protein loading.

Journal: Frontiers in Cellular Neuroscience

Article Title: Mitochondrial DNMT3A and DNA methylation in skeletal muscle and CNS of transgenic mouse models of ALS

doi: 10.3389/fncel.2013.00279

Figure Lengend Snippet: Dnmt3a levels are reduced in hSOD1 tg mouse models of ALS . (A) Western blot for Dnmt3a in leg skeletal muscle mitochondria (100 μg/lane) in non-conditional tg mice expressing hSOD1-G37R or hSOD1-wildtype constructs and conditional (muscle-restricted) tg mice expressing hSOD1 mus -G37R, -G93A, or -wildtype constructs compared to age-matched non-tg littermates. Ponceau S staining of membrane shows protein loading. (B) Graph showing the relative levels of Dnmt3a immunoreactivity determined by immunoblotting of pure mitochondria isolated from hSOD1 tg mouse leg skeletal muscle. Values are mean ± SD. Significant differences from non-tg control are indicated by single ( p < 0.05) or double ( p < 0.01) asterisks. (C) Western blot for Dnmt3a in spinal cord mitochondria fractions (50 μg/lane) in conditional (muscle-restricted) tg mice expressing hSOD1 mus -G37R, -G93A, or -wildtype constructs compared to age-matched non-tg littermates. Ponceau S staining of membrane shows protein loading. (D) Graph showing the relative levels of Dnmt3a immunoreactivity in hSOD1 mus tg mouse spinal cord. Values are mean ± SD. Significant differences from non-tg control are indicated by an asterisk ( p < 0.05). The results have been replicated in at least 3 different experiments. (E) Western blot for Dnmt3a in crude fractions of skeletal muscle and spinal cord of conditional (muscle-restricted) tg mice expressing hSOD1 mus -G37R or -wildtype constructs compared to age-matched non-tg littermates. Ponceau S staining of membrane shows protein loading.

Article Snippet: The membranes were stained with Ponceau S (Sigma) to determine transfer efficiency, destained, blocked with 1% BSA/0.05% Tween 20/TBS and incubated with primary antibodies: rabbit polyclonal anti-cytochrome P450 reductase (Stressgen) at 1:1000; mouse monoclonal anti-actin (Chemicon) at 1:1000; mouse monoclonal anti-porin (Mitosciences) at 1:3000; rabbit polyclonal anti-lamin (Millipore) at 1:500; sheep polyclonal anti-catalase (Biodesign International) at 1:2000; mouse anti-cyclophilin D (Mitosciences) at 1:3000; mouse monoclonal anti-complex V (Molecular Probes Invitrogen) at 1:10,000; rabbit polyclonal and mouse monoclonal anti-Dnmt3a (Cell Signaling, Abgent, and Alexis) at 1:100–1:500, rabbit polyclonal and mouse monoclonal anti-Dnmt1 (Bethyl, Novus, and Alexis) at 1:500–1:5000, and rabbit polyclonal antibody to Dnmt3b (Abcam) at 1:250.

Techniques: Western Blot, Expressing, Construct, Staining, Isolation

Cellular localizations of Dnmt3a and 5-methylcytosine (5mC) in wildtype young adult mouse spinal cord and testes . (A) In 6–8 weeks old non-tg mice, Dnmt3a is present in the nucleus and in the cytoplasm of neurons in spinal cord. Extranuclear cytoplasmic Dnmt3a immunoreactivity (green) is localized diffusely and in discrete bodies (arrows). These discrete bodies also contain 5mC (red, arrows). Hoechst staining was used to identify cell nuclei. (B) Cytoplasmic 5mC is localized to mitochondria in neurons. Some mitochondria, identified by SOD2 immunoreactivity (green, arrows), in spinal cord colocalize with 5mC immunoreactivity (red, arrows). Many mitochondria also do not contain 5mC. The SOD2- and 5mC-positive bodies tend to be larger than normal mitochondria (white arrows). (C) 5mC is present in autophagosomes. Mouse spinal cord sections were immunostained for an autophagosome marker LC3A (green) and 5mC (red). Extranuclear 5mC immunoreactivity has a high colocalization with LC3A in autophagosomes (arrows). (D,E) Immunofluorescent localization of Dnmt3a (red) and complex V (green) in mouse testis. Cell nuclei are blue. Dnmt3a immunoreactivity (red) is enriched in cells in the seminiferous tubules of testes. The boxed area in D is represented at higher magnification in E which shows that Dnmt3a immunoreactivity (red) partly colocalizes (seen as yellow-orange) with mitochondria (green). Scale bars: 30 μm (D) ; 15 μm (E) . (F) Immunofluorescence for 5mC (red) and SOD2 (green) in mouse testes. Cell nuclei are blue. 5mC immunoreactivity (red) partly colocalizes (seen as yellow-orange) with mitochondria (green). Mature spermatozoa with scant mitochondria are seen at left in seminiferous tubule lumen (L). Scale bar = 30 μm.

Journal: Frontiers in Cellular Neuroscience

Article Title: Mitochondrial DNMT3A and DNA methylation in skeletal muscle and CNS of transgenic mouse models of ALS

doi: 10.3389/fncel.2013.00279

Figure Lengend Snippet: Cellular localizations of Dnmt3a and 5-methylcytosine (5mC) in wildtype young adult mouse spinal cord and testes . (A) In 6–8 weeks old non-tg mice, Dnmt3a is present in the nucleus and in the cytoplasm of neurons in spinal cord. Extranuclear cytoplasmic Dnmt3a immunoreactivity (green) is localized diffusely and in discrete bodies (arrows). These discrete bodies also contain 5mC (red, arrows). Hoechst staining was used to identify cell nuclei. (B) Cytoplasmic 5mC is localized to mitochondria in neurons. Some mitochondria, identified by SOD2 immunoreactivity (green, arrows), in spinal cord colocalize with 5mC immunoreactivity (red, arrows). Many mitochondria also do not contain 5mC. The SOD2- and 5mC-positive bodies tend to be larger than normal mitochondria (white arrows). (C) 5mC is present in autophagosomes. Mouse spinal cord sections were immunostained for an autophagosome marker LC3A (green) and 5mC (red). Extranuclear 5mC immunoreactivity has a high colocalization with LC3A in autophagosomes (arrows). (D,E) Immunofluorescent localization of Dnmt3a (red) and complex V (green) in mouse testis. Cell nuclei are blue. Dnmt3a immunoreactivity (red) is enriched in cells in the seminiferous tubules of testes. The boxed area in D is represented at higher magnification in E which shows that Dnmt3a immunoreactivity (red) partly colocalizes (seen as yellow-orange) with mitochondria (green). Scale bars: 30 μm (D) ; 15 μm (E) . (F) Immunofluorescence for 5mC (red) and SOD2 (green) in mouse testes. Cell nuclei are blue. 5mC immunoreactivity (red) partly colocalizes (seen as yellow-orange) with mitochondria (green). Mature spermatozoa with scant mitochondria are seen at left in seminiferous tubule lumen (L). Scale bar = 30 μm.

Article Snippet: The membranes were stained with Ponceau S (Sigma) to determine transfer efficiency, destained, blocked with 1% BSA/0.05% Tween 20/TBS and incubated with primary antibodies: rabbit polyclonal anti-cytochrome P450 reductase (Stressgen) at 1:1000; mouse monoclonal anti-actin (Chemicon) at 1:1000; mouse monoclonal anti-porin (Mitosciences) at 1:3000; rabbit polyclonal anti-lamin (Millipore) at 1:500; sheep polyclonal anti-catalase (Biodesign International) at 1:2000; mouse anti-cyclophilin D (Mitosciences) at 1:3000; mouse monoclonal anti-complex V (Molecular Probes Invitrogen) at 1:10,000; rabbit polyclonal and mouse monoclonal anti-Dnmt3a (Cell Signaling, Abgent, and Alexis) at 1:100–1:500, rabbit polyclonal and mouse monoclonal anti-Dnmt1 (Bethyl, Novus, and Alexis) at 1:500–1:5000, and rabbit polyclonal antibody to Dnmt3b (Abcam) at 1:250.

Techniques: Staining, Marker, Immunofluorescence

Immunofluorescent localizations of Dnmt3a, mitochondria, and 5-methylcytosine (5mC) in hSOD1 mus tg mouse skeletal muscle and spinal cord . (A,B) In non-tg mouse skeletal muscle (biceps femoris), Dnmt3a (red) immunoreactivity can be found diffusely in the sarcoplasm and associated with mitochondria (colocalization is seen as yellow) identified by complex V immunoreactivity (green). Dnmt3a/complex V colocalization occur prominently in subsarcolemmal mitochondria in non-tg mice (A, open arrows) and in interfibrillar mitochondria (A , hatched arrow ) . In hSOD1 mus mouse hindleg skeletal muscle, Dnmt3a (red) immunoreactivity and mitochondrial complex V immunoreactivity (green) are markedly attenuated. Scale bars = 9 μm (A) , 15 μm (B) . (C,D) . Myofiber perinuclear colocalization of Dnmt3a and 5mC is intensified in hSOD1 mus tg mice. In non-tg mouse skeletal muscle (biceps femoris), Dnmt3a (red) immunoreactivity can be found clustered around peripheral myonuclei (C , hatched arrows ) and generally has little colocalization with 5mC. In hSOD1 mus mouse skeletal muscle Dnmt3a (red) and 5mC (green) immunoreactivities have prominent perinuclear colocalizations (D , hatched arrows, yellow-orange ) . Scale bars = 20 μm (C,D) . (E,F) In ~17 months old non-tg mice, Dnmt3a immunoreactivity (red) is present in the nucleus and, more prominently, in the cytoplasm of spinal cord motor neurons (E,G , open arrows ) . Cytoplasmic Dnmt3a immunoreactivity (red) is localized in discreet particles in motor neurons (E , G , arrows ) . Many of these cytoplasmic particles colocalize (seen as yellow) with complex V immunoreactivity (E , green ) , and thus are mitochondria, and with 5mC (G , green, arrow ) and thus contain methylated mtDNA. Scale bar in (E) ( same for F–H) = 6 μm. In age-matched hSOD1 mus tg mice, remaining spinal motor neurons (F , open arrow ) show intensified mitochondrial immunoreactivity for complex V (green) and the Dnmt3a immunoreactivity largely is colocalized with complex V (F) and 5mC (H) and is aggregated in the cytoplasm.

Journal: Frontiers in Cellular Neuroscience

Article Title: Mitochondrial DNMT3A and DNA methylation in skeletal muscle and CNS of transgenic mouse models of ALS

doi: 10.3389/fncel.2013.00279

Figure Lengend Snippet: Immunofluorescent localizations of Dnmt3a, mitochondria, and 5-methylcytosine (5mC) in hSOD1 mus tg mouse skeletal muscle and spinal cord . (A,B) In non-tg mouse skeletal muscle (biceps femoris), Dnmt3a (red) immunoreactivity can be found diffusely in the sarcoplasm and associated with mitochondria (colocalization is seen as yellow) identified by complex V immunoreactivity (green). Dnmt3a/complex V colocalization occur prominently in subsarcolemmal mitochondria in non-tg mice (A, open arrows) and in interfibrillar mitochondria (A , hatched arrow ) . In hSOD1 mus mouse hindleg skeletal muscle, Dnmt3a (red) immunoreactivity and mitochondrial complex V immunoreactivity (green) are markedly attenuated. Scale bars = 9 μm (A) , 15 μm (B) . (C,D) . Myofiber perinuclear colocalization of Dnmt3a and 5mC is intensified in hSOD1 mus tg mice. In non-tg mouse skeletal muscle (biceps femoris), Dnmt3a (red) immunoreactivity can be found clustered around peripheral myonuclei (C , hatched arrows ) and generally has little colocalization with 5mC. In hSOD1 mus mouse skeletal muscle Dnmt3a (red) and 5mC (green) immunoreactivities have prominent perinuclear colocalizations (D , hatched arrows, yellow-orange ) . Scale bars = 20 μm (C,D) . (E,F) In ~17 months old non-tg mice, Dnmt3a immunoreactivity (red) is present in the nucleus and, more prominently, in the cytoplasm of spinal cord motor neurons (E,G , open arrows ) . Cytoplasmic Dnmt3a immunoreactivity (red) is localized in discreet particles in motor neurons (E , G , arrows ) . Many of these cytoplasmic particles colocalize (seen as yellow) with complex V immunoreactivity (E , green ) , and thus are mitochondria, and with 5mC (G , green, arrow ) and thus contain methylated mtDNA. Scale bar in (E) ( same for F–H) = 6 μm. In age-matched hSOD1 mus tg mice, remaining spinal motor neurons (F , open arrow ) show intensified mitochondrial immunoreactivity for complex V (green) and the Dnmt3a immunoreactivity largely is colocalized with complex V (F) and 5mC (H) and is aggregated in the cytoplasm.

Article Snippet: The membranes were stained with Ponceau S (Sigma) to determine transfer efficiency, destained, blocked with 1% BSA/0.05% Tween 20/TBS and incubated with primary antibodies: rabbit polyclonal anti-cytochrome P450 reductase (Stressgen) at 1:1000; mouse monoclonal anti-actin (Chemicon) at 1:1000; mouse monoclonal anti-porin (Mitosciences) at 1:3000; rabbit polyclonal anti-lamin (Millipore) at 1:500; sheep polyclonal anti-catalase (Biodesign International) at 1:2000; mouse anti-cyclophilin D (Mitosciences) at 1:3000; mouse monoclonal anti-complex V (Molecular Probes Invitrogen) at 1:10,000; rabbit polyclonal and mouse monoclonal anti-Dnmt3a (Cell Signaling, Abgent, and Alexis) at 1:100–1:500, rabbit polyclonal and mouse monoclonal anti-Dnmt1 (Bethyl, Novus, and Alexis) at 1:500–1:5000, and rabbit polyclonal antibody to Dnmt3b (Abcam) at 1:250.

Techniques: Methylation

Correlation analysis between plasma homocysteine levels (μmol/L) and hepatic Dnmt3a mRNA levels (AU). Male rats are represented by squares and females by crosses.

Journal: International Journal of Molecular Sciences

Article Title: Maternal Methyl Donors Supplementation during Lactation Prevents the Hyperhomocysteinemia Induced by a High-Fat-Sucrose Intake by Dams

doi: 10.3390/ijms141224422

Figure Lengend Snippet: Correlation analysis between plasma homocysteine levels (μmol/L) and hepatic Dnmt3a mRNA levels (AU). Male rats are represented by squares and females by crosses.

Article Snippet: Quantitative real-time PCR ( n = 6 animals from 5–6 L per maternal dietary group) was performed by triplicate using ABI PRISM 7900 HT Fast real-time PCR system (Applied Biosystems, Austin, TX, USA) and Taqman primers (Applied Biosystems, Austin, TX, USA) for Dnmt1 (Rn00709664_m1*), Dnmt3a (Rn01027162_g1*) and Dnmt3b (Rn01536419_m1).

Techniques: Clinical Proteomics

Association analysis between polymorphisms of DNA methyltransferase genes and risk of grade ≥ 2 radiation-induced fibrosis (LENT-SOMA scale) in breast cancer patients

Journal: Cancer Research and Treatment : Official Journal of Korean Cancer Association

Article Title: DNA Methyltransferase Gene Polymorphisms for Prediction of Radiation-Induced Skin Fibrosis after Treatment of Breast Cancer: A Multifactorial Genetic Approach

doi: 10.4143/crt.2016.256

Figure Lengend Snippet: Association analysis between polymorphisms of DNA methyltransferase genes and risk of grade ≥ 2 radiation-induced fibrosis (LENT-SOMA scale) in breast cancer patients

Article Snippet: Determination of SNPs was conducted on genomic DNA by real-time polymerase chain reaction (PCR) using the following TaqMan Pre-Designed SNP Genotyping assays (Applied Biosystems, Milan, Italy): C_27838930_10 ( DNMT1 rs2228611); C_8722920_10 ( DNMT3A rs1550117); C_7863728_10 ( DNMT3A rs7581217); C_16013055_10 ( DNMT3B rs2424908); and C_16269889_10 ( XRCC1 rs2682585).

Techniques: Significance Assay

(A) MSP showed that the methylation level in the pre-miR-145 promoter of SKOV3 and 3AO cells was decreased by 20(S)-Rg3. M: methylated product, U: unmethylated product. Fisher exact test. (B) Western blot analysis indicated that DNMT3A rather than DNMT3B and DNMT1 was significantly decreased in SKOV3 and 3AO cells treated by 20(S)-Rg3. (C) qRT-PCR showed that transfection of DNMT3A plasmid rescued DNMT3A level in 20(S)-Rg3-treated cells. t test. (D) qRT-PCR showed overexpression of DNMT3A reversed 20(S)-Rg3-induced miR-145. (E) MSP showed the methylation level in the pre-miR-145 promoter was increased in cells treated with both 20(S)-Rg3 and DNMT3A plasmid compared to that in cells treated with 20(S)-Rg3 alone. M: methylated product, U: unmethylated product. Fisher exact test. (F) Western blot analysis showed that restoration of DNMT3A reversed 20(S)-Rg3-induced E-cadherin increase, N-cadherin and vimentin decrease. (G) In vitro migration assay showed that transfection of DNMT3A plasmid stimulated motility and invasion of 20(S)-Rg3-treated cells (200×). t test. All experiments were performed in triplicate and data were showed as means ± SE.

Journal: Oncotarget

Article Title: 20(S)-Rg3 blocked epithelial-mesenchymal transition through DNMT3A/miR-145/FSCN1 in ovarian cancer

doi: 10.18632/oncotarget.18482

Figure Lengend Snippet: (A) MSP showed that the methylation level in the pre-miR-145 promoter of SKOV3 and 3AO cells was decreased by 20(S)-Rg3. M: methylated product, U: unmethylated product. Fisher exact test. (B) Western blot analysis indicated that DNMT3A rather than DNMT3B and DNMT1 was significantly decreased in SKOV3 and 3AO cells treated by 20(S)-Rg3. (C) qRT-PCR showed that transfection of DNMT3A plasmid rescued DNMT3A level in 20(S)-Rg3-treated cells. t test. (D) qRT-PCR showed overexpression of DNMT3A reversed 20(S)-Rg3-induced miR-145. (E) MSP showed the methylation level in the pre-miR-145 promoter was increased in cells treated with both 20(S)-Rg3 and DNMT3A plasmid compared to that in cells treated with 20(S)-Rg3 alone. M: methylated product, U: unmethylated product. Fisher exact test. (F) Western blot analysis showed that restoration of DNMT3A reversed 20(S)-Rg3-induced E-cadherin increase, N-cadherin and vimentin decrease. (G) In vitro migration assay showed that transfection of DNMT3A plasmid stimulated motility and invasion of 20(S)-Rg3-treated cells (200×). t test. All experiments were performed in triplicate and data were showed as means ± SE.

Article Snippet: Antibodies including DNMT3A, DNMT3B, E-cadherin, N-cadherin, vimentin, and β-actin were from Cell Signaling Technology (Beverly, MA), FSCN1 was from Abcam (Cambridge, MA, USA), and DNMT1 was from Active Motif (Carlsbad, CA, USA).

Techniques: Methylation, Western Blot, Quantitative RT-PCR, Transfection, Plasmid Preparation, Over Expression, In Vitro, Migration

(A) qRT-PCR showed that ectopic expression of miR-145 had negligible effect on DNMT3A expression in DNMT3A-overexpressed cells. (B) qRT-PCR showed that DNMT3A overexpression downregulated miR-145 expression which was recovered by mimic145 transfection. (C) Western blot analysis indicated that E-cadherin decrease, N-cadherin and vimentin increase caused by DNMT3A were reversed by concomitant miR-145 overexpression. (D) In vitro migration and invasion assays indicated that overexpression of miR-145 largely attenuated DNMT3A promotion on the migration and invasion (200×). All experiments were performed in triplicate and datawere showed as means ± SE. t-test.

Journal: Oncotarget

Article Title: 20(S)-Rg3 blocked epithelial-mesenchymal transition through DNMT3A/miR-145/FSCN1 in ovarian cancer

doi: 10.18632/oncotarget.18482

Figure Lengend Snippet: (A) qRT-PCR showed that ectopic expression of miR-145 had negligible effect on DNMT3A expression in DNMT3A-overexpressed cells. (B) qRT-PCR showed that DNMT3A overexpression downregulated miR-145 expression which was recovered by mimic145 transfection. (C) Western blot analysis indicated that E-cadherin decrease, N-cadherin and vimentin increase caused by DNMT3A were reversed by concomitant miR-145 overexpression. (D) In vitro migration and invasion assays indicated that overexpression of miR-145 largely attenuated DNMT3A promotion on the migration and invasion (200×). All experiments were performed in triplicate and datawere showed as means ± SE. t-test.

Article Snippet: Antibodies including DNMT3A, DNMT3B, E-cadherin, N-cadherin, vimentin, and β-actin were from Cell Signaling Technology (Beverly, MA), FSCN1 was from Abcam (Cambridge, MA, USA), and DNMT1 was from Active Motif (Carlsbad, CA, USA).

Techniques: Quantitative RT-PCR, Expressing, Over Expression, Transfection, Western Blot, In Vitro, Migration

20(S)-Rg3 down-regulated DNMT3A to demethylate pre-miR-145 and thus upregulated mature miR-145 that targeted FSCN1 and finally blocked EMT to attenuate cell migration and invasion.

Journal: Oncotarget

Article Title: 20(S)-Rg3 blocked epithelial-mesenchymal transition through DNMT3A/miR-145/FSCN1 in ovarian cancer

doi: 10.18632/oncotarget.18482

Figure Lengend Snippet: 20(S)-Rg3 down-regulated DNMT3A to demethylate pre-miR-145 and thus upregulated mature miR-145 that targeted FSCN1 and finally blocked EMT to attenuate cell migration and invasion.

Article Snippet: Antibodies including DNMT3A, DNMT3B, E-cadherin, N-cadherin, vimentin, and β-actin were from Cell Signaling Technology (Beverly, MA), FSCN1 was from Abcam (Cambridge, MA, USA), and DNMT1 was from Active Motif (Carlsbad, CA, USA).

Techniques: Migration