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:

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

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

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

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

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

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