dnmt1  (New England Biolabs)


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    Structured Review

    New England Biolabs dnmt1
    hUHRF1 recruits G9a and other chromatin modification enzymes to p21 promoter. ( A ) Linearity of PCR amplification using primer sets for proximal (−385 to −240) and distal (−4164 to −3959) regions of p21 promoter with increasing amount of input DNA. ( B ) ChIP analysis of p21 promoter after KD of hUHRF1. HeLa cells were transfected with either control siRNA (CTL KD) or hUHRF1 siRNA (hUHRF1 KD). Using the chromatin isolated from the KD cells, ChIP was performed to detect the proteins or histone modification as indicated at the top of the panel. 5% input is shown. ( C ) Quantitative ChIP analysis for relative p21 promoter occupancy of hUHRF1, G9a and dimethylated H3K9 (H3K9me2) after KD of hUHRF1 or G9a. Q-PCR data of each group were normalized to its input as % input. The relative p21 promoter occupancy of hUHRF1 or G9a KD samples represents the fold change in percentage input over that of the CTL KD. Error bars indicate standard deviation of three independent experiments. ( D ) ChIP analysis of p21 promoter after KD of <t>DNMT1.</t> HeLa cells were transfected with either control siRNA (CTL KD) or DNMT1 siRNA (DNMT1 KD). CHIP was performed as described in (B) and 5% input is shown.
    Dnmt1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    1) Product Images from "UHRF1 binds G9a and participates in p21 transcriptional regulation in mammalian cells"

    Article Title: UHRF1 binds G9a and participates in p21 transcriptional regulation in mammalian cells

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gkn961

    hUHRF1 recruits G9a and other chromatin modification enzymes to p21 promoter. ( A ) Linearity of PCR amplification using primer sets for proximal (−385 to −240) and distal (−4164 to −3959) regions of p21 promoter with increasing amount of input DNA. ( B ) ChIP analysis of p21 promoter after KD of hUHRF1. HeLa cells were transfected with either control siRNA (CTL KD) or hUHRF1 siRNA (hUHRF1 KD). Using the chromatin isolated from the KD cells, ChIP was performed to detect the proteins or histone modification as indicated at the top of the panel. 5% input is shown. ( C ) Quantitative ChIP analysis for relative p21 promoter occupancy of hUHRF1, G9a and dimethylated H3K9 (H3K9me2) after KD of hUHRF1 or G9a. Q-PCR data of each group were normalized to its input as % input. The relative p21 promoter occupancy of hUHRF1 or G9a KD samples represents the fold change in percentage input over that of the CTL KD. Error bars indicate standard deviation of three independent experiments. ( D ) ChIP analysis of p21 promoter after KD of DNMT1. HeLa cells were transfected with either control siRNA (CTL KD) or DNMT1 siRNA (DNMT1 KD). CHIP was performed as described in (B) and 5% input is shown.
    Figure Legend Snippet: hUHRF1 recruits G9a and other chromatin modification enzymes to p21 promoter. ( A ) Linearity of PCR amplification using primer sets for proximal (−385 to −240) and distal (−4164 to −3959) regions of p21 promoter with increasing amount of input DNA. ( B ) ChIP analysis of p21 promoter after KD of hUHRF1. HeLa cells were transfected with either control siRNA (CTL KD) or hUHRF1 siRNA (hUHRF1 KD). Using the chromatin isolated from the KD cells, ChIP was performed to detect the proteins or histone modification as indicated at the top of the panel. 5% input is shown. ( C ) Quantitative ChIP analysis for relative p21 promoter occupancy of hUHRF1, G9a and dimethylated H3K9 (H3K9me2) after KD of hUHRF1 or G9a. Q-PCR data of each group were normalized to its input as % input. The relative p21 promoter occupancy of hUHRF1 or G9a KD samples represents the fold change in percentage input over that of the CTL KD. Error bars indicate standard deviation of three independent experiments. ( D ) ChIP analysis of p21 promoter after KD of DNMT1. HeLa cells were transfected with either control siRNA (CTL KD) or DNMT1 siRNA (DNMT1 KD). CHIP was performed as described in (B) and 5% input is shown.

    Techniques Used: Modification, Polymerase Chain Reaction, Amplification, Chromatin Immunoprecipitation, Transfection, CTL Assay, Isolation, Standard Deviation

    2) Product Images from "Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription *"

    Article Title: Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription *

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M601155200

    Ectopic DNMT1 and DNMT3B, but not DNMT3A, inhibit rDNA promoter activity irrespective of its methylation status A , HeLa cells were transiently transfected with DNMT1, DNMT3A, or DNMT3B expression vectors. Whole cell extracts from these cells were subjected to Western blot analysis with anti-FLAG M2 antibody. B , HeLa cells were transfected with either mock methylated pHrD-IRES-Luc ( Unmethylated ) or M.HhaI-methylated pHrD-IRES-Luc ( Methylated ) along with 4 μ g of the empty vector or pcDNMT1, pcDNMT3A, or pcDNMT3B. After 48 h firefly luciferase activity was measured in the cell extracts. Results are represented as rDNA promoter activity upon overexpression of different DNMTs with empty vector-transfected pHrD-IRES-Luc activity as 100.
    Figure Legend Snippet: Ectopic DNMT1 and DNMT3B, but not DNMT3A, inhibit rDNA promoter activity irrespective of its methylation status A , HeLa cells were transiently transfected with DNMT1, DNMT3A, or DNMT3B expression vectors. Whole cell extracts from these cells were subjected to Western blot analysis with anti-FLAG M2 antibody. B , HeLa cells were transfected with either mock methylated pHrD-IRES-Luc ( Unmethylated ) or M.HhaI-methylated pHrD-IRES-Luc ( Methylated ) along with 4 μ g of the empty vector or pcDNMT1, pcDNMT3A, or pcDNMT3B. After 48 h firefly luciferase activity was measured in the cell extracts. Results are represented as rDNA promoter activity upon overexpression of different DNMTs with empty vector-transfected pHrD-IRES-Luc activity as 100.

    Techniques Used: Activity Assay, Methylation, Transfection, Expressing, Western Blot, Plasmid Preparation, Luciferase, Over Expression

    A , all three DNMTs localize in the nucleolus. HeLa cells were stained with TRITC-tagged mouse monoclonal antibody against nucleolin and with fluorescein isothiocyanate-tagged rabbit polyclonal antibody against DNMT1, 3A, -3B, or UBF ( panels a–t ). In a separate set, cells were stained with fluorescein isothiocyanate-tagged rabbit polyclonal antibody against UBF and TRITC-tagged mouse monoclonal antibody against DNMT1 ( panels u–y ). All five sets of cells were also stained with 4′,6-diamidino-2-phenylindole and visualized under a fluorescence microscope. B , all three DNMTs co-fractionated with nucleolin in the nucleolar fraction: nucleolar extract and nuclear extract (nucleolus and nucleoplasm) from HeLa cells (250 μ g) were subjected to Western blot analysis with antibodies against nucleolin, RNA polymerase II, DNMT1, -3A, and -3B. C , DNMT1, -3B, and -3A are associated with methylated rDNA promoter. Formaldehyde cross-linked chromatin was precleared and immunoprecipitated overnight with antisera specific for DNMT1, DNMT3A, DNMT3B, UBF, or preimmune sera. The immune complexes were precipitated by protein A/G beads, washed with different buffers (detailed under “Experimental Procedures”), eluted, and un-cross-linked. DNAs pulled down by different antibodies as well as input DNA were divided into three identical fractions that were either mock digested or digested with HpaII or MspI. An aliquot of each digestion product was subjected to real time PCR with primers specific for rDNA promoter. Association of different DNMTs and UBF with the rDNA promoter was analyzed using a standard curve generated by serial dilution of the undigested input DNA. Association with methylated promoter equals HpaII signal in ChIP DNA/HpaII signal in input (1:300 dilution). Association with unmethylated promoter corresponds to the signal in undigested minus signal in HpaII-digested ChIP DNA/Input signal in undigested minus HpaII-digested DNA (1:300 dilution). U and M indicate methylated and unmethylated rDNA promoters, respectively.
    Figure Legend Snippet: A , all three DNMTs localize in the nucleolus. HeLa cells were stained with TRITC-tagged mouse monoclonal antibody against nucleolin and with fluorescein isothiocyanate-tagged rabbit polyclonal antibody against DNMT1, 3A, -3B, or UBF ( panels a–t ). In a separate set, cells were stained with fluorescein isothiocyanate-tagged rabbit polyclonal antibody against UBF and TRITC-tagged mouse monoclonal antibody against DNMT1 ( panels u–y ). All five sets of cells were also stained with 4′,6-diamidino-2-phenylindole and visualized under a fluorescence microscope. B , all three DNMTs co-fractionated with nucleolin in the nucleolar fraction: nucleolar extract and nuclear extract (nucleolus and nucleoplasm) from HeLa cells (250 μ g) were subjected to Western blot analysis with antibodies against nucleolin, RNA polymerase II, DNMT1, -3A, and -3B. C , DNMT1, -3B, and -3A are associated with methylated rDNA promoter. Formaldehyde cross-linked chromatin was precleared and immunoprecipitated overnight with antisera specific for DNMT1, DNMT3A, DNMT3B, UBF, or preimmune sera. The immune complexes were precipitated by protein A/G beads, washed with different buffers (detailed under “Experimental Procedures”), eluted, and un-cross-linked. DNAs pulled down by different antibodies as well as input DNA were divided into three identical fractions that were either mock digested or digested with HpaII or MspI. An aliquot of each digestion product was subjected to real time PCR with primers specific for rDNA promoter. Association of different DNMTs and UBF with the rDNA promoter was analyzed using a standard curve generated by serial dilution of the undigested input DNA. Association with methylated promoter equals HpaII signal in ChIP DNA/HpaII signal in input (1:300 dilution). Association with unmethylated promoter corresponds to the signal in undigested minus signal in HpaII-digested ChIP DNA/Input signal in undigested minus HpaII-digested DNA (1:300 dilution). U and M indicate methylated and unmethylated rDNA promoters, respectively.

    Techniques Used: Staining, Fluorescence, Microscopy, Western Blot, Methylation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Generated, Serial Dilution, Chromatin Immunoprecipitation

    Catalytic domain of DNMT1 is necessary to inhibit unmethylated rDNA promoter that is de novo methylated by ectopic DNMT1 A , schematic diagram of different deletion/point mutants of DNMT1 used in transient transfection studies. B , HeLa cells were transfected with mock methylated pHrD-IRES-Luc and empty or expression vectors for different deletion mutants of DNMT1 (Δ CAT , catalytic domain deleted; CS , cysteine to serine mutation at PCQ motif; Δ NLS , NH 2 -terminal domain deleted including the nuclear localization signal). After 48 h firefly luciferase activity was measured in cell extracts. Results are represented as rDNA promoter activity upon overexpression of different DNMT1 mutants. One hundred percent activity represents empty vector-transfected pHrD-IRES-Luc activity. C , HeLa cells were cotransfected with mock-methylated pHrD-IRES-Luc and either empty vector or DNMT1 expression vector. The pHrD-IRES-Luc plasmid was isolated from the transfected cells 48 h post-transfection and digested with HpaII or MspI. The rDNA promoter was amplified from mock digested ( U ), HpaII ( H ), or MspI ( M ) digested plasmid using a vector-specific and a rDNA promoter-specific primer and resolved on 1% agarose gel.
    Figure Legend Snippet: Catalytic domain of DNMT1 is necessary to inhibit unmethylated rDNA promoter that is de novo methylated by ectopic DNMT1 A , schematic diagram of different deletion/point mutants of DNMT1 used in transient transfection studies. B , HeLa cells were transfected with mock methylated pHrD-IRES-Luc and empty or expression vectors for different deletion mutants of DNMT1 (Δ CAT , catalytic domain deleted; CS , cysteine to serine mutation at PCQ motif; Δ NLS , NH 2 -terminal domain deleted including the nuclear localization signal). After 48 h firefly luciferase activity was measured in cell extracts. Results are represented as rDNA promoter activity upon overexpression of different DNMT1 mutants. One hundred percent activity represents empty vector-transfected pHrD-IRES-Luc activity. C , HeLa cells were cotransfected with mock-methylated pHrD-IRES-Luc and either empty vector or DNMT1 expression vector. The pHrD-IRES-Luc plasmid was isolated from the transfected cells 48 h post-transfection and digested with HpaII or MspI. The rDNA promoter was amplified from mock digested ( U ), HpaII ( H ), or MspI ( M ) digested plasmid using a vector-specific and a rDNA promoter-specific primer and resolved on 1% agarose gel.

    Techniques Used: Methylation, Transfection, Expressing, Mutagenesis, Luciferase, Activity Assay, Over Expression, Plasmid Preparation, Isolation, Amplification, Agarose Gel Electrophoresis

    DNMT1 and -3B synergistically maintain rDNA promoter methylation A , whole cell extracts from HCT116 ( WT ), DNMT1 −/− ( MT1KO ), DNMT3B −/− ( 3BKO ), and DNMT1 −/− /DNMT3B −/− ( DKO ) cells were separated by SDS-PAGE and Western blot analysis was performed with specific antibodies. B , schematic presentation of the rDNA promoter region used as probe for Southern blot analysis. C , genomic DNA from the cells were either mock-digested ( U , Uncut), and digested with Hpall ( H ) or MspI ( M ). The digested products were separated on a 0.8% agarose gel and stained with ethidium bromide. D , DNA was transferred to nylon membrane and probed with a 32 P-labled rDNA promoter probe. E , rDNA, GAPDH, and albumin promoters were amplified from mock-digested ( U , Uncut), Hpall ( H ), or MspI ( M ) digested genomic DNA from W T HCT116, MT1KO, 3BKO, and DKO cells and resolved on 1.5% agarose gel. F , quantitative analysis of the PCR amplified rDNA products. G , total RNA isolated from the cells was resolved on a 1.0% agarose/formaldehyde gel, transferred to nylon membrane, and probed with the 91-bp pre-RNA probe. H , total RNA from the cells was treated with RNase-free DNase I, reverse transcribed, and subjected to real time PCR using 47 S rRNA-specific primers. Data presented was normalized to 18 S rRNA. RNA without reverse transcription did not generate PCR product.
    Figure Legend Snippet: DNMT1 and -3B synergistically maintain rDNA promoter methylation A , whole cell extracts from HCT116 ( WT ), DNMT1 −/− ( MT1KO ), DNMT3B −/− ( 3BKO ), and DNMT1 −/− /DNMT3B −/− ( DKO ) cells were separated by SDS-PAGE and Western blot analysis was performed with specific antibodies. B , schematic presentation of the rDNA promoter region used as probe for Southern blot analysis. C , genomic DNA from the cells were either mock-digested ( U , Uncut), and digested with Hpall ( H ) or MspI ( M ). The digested products were separated on a 0.8% agarose gel and stained with ethidium bromide. D , DNA was transferred to nylon membrane and probed with a 32 P-labled rDNA promoter probe. E , rDNA, GAPDH, and albumin promoters were amplified from mock-digested ( U , Uncut), Hpall ( H ), or MspI ( M ) digested genomic DNA from W T HCT116, MT1KO, 3BKO, and DKO cells and resolved on 1.5% agarose gel. F , quantitative analysis of the PCR amplified rDNA products. G , total RNA isolated from the cells was resolved on a 1.0% agarose/formaldehyde gel, transferred to nylon membrane, and probed with the 91-bp pre-RNA probe. H , total RNA from the cells was treated with RNase-free DNase I, reverse transcribed, and subjected to real time PCR using 47 S rRNA-specific primers. Data presented was normalized to 18 S rRNA. RNA without reverse transcription did not generate PCR product.

    Techniques Used: Methylation, SDS Page, Western Blot, Southern Blot, Agarose Gel Electrophoresis, Staining, Amplification, Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction

    3) Product Images from "Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription *"

    Article Title: Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription *

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M601155200

    Ectopic DNMT1 and DNMT3B, but not DNMT3A, inhibit rDNA promoter activity irrespective of its methylation status A , HeLa cells were transiently transfected with DNMT1, DNMT3A, or DNMT3B expression vectors. Whole cell extracts from these cells were subjected to Western blot analysis with anti-FLAG M2 antibody. B , HeLa cells were transfected with either mock methylated pHrD-IRES-Luc ( Unmethylated ) or M.HhaI-methylated pHrD-IRES-Luc ( Methylated ) along with 4 μ g of the empty vector or pcDNMT1, pcDNMT3A, or pcDNMT3B. After 48 h firefly luciferase activity was measured in the cell extracts. Results are represented as rDNA promoter activity upon overexpression of different DNMTs with empty vector-transfected pHrD-IRES-Luc activity as 100.
    Figure Legend Snippet: Ectopic DNMT1 and DNMT3B, but not DNMT3A, inhibit rDNA promoter activity irrespective of its methylation status A , HeLa cells were transiently transfected with DNMT1, DNMT3A, or DNMT3B expression vectors. Whole cell extracts from these cells were subjected to Western blot analysis with anti-FLAG M2 antibody. B , HeLa cells were transfected with either mock methylated pHrD-IRES-Luc ( Unmethylated ) or M.HhaI-methylated pHrD-IRES-Luc ( Methylated ) along with 4 μ g of the empty vector or pcDNMT1, pcDNMT3A, or pcDNMT3B. After 48 h firefly luciferase activity was measured in the cell extracts. Results are represented as rDNA promoter activity upon overexpression of different DNMTs with empty vector-transfected pHrD-IRES-Luc activity as 100.

    Techniques Used: Activity Assay, Methylation, Transfection, Expressing, Western Blot, Plasmid Preparation, Luciferase, Over Expression

    A , all three DNMTs localize in the nucleolus. HeLa cells were stained with TRITC-tagged mouse monoclonal antibody against nucleolin and with fluorescein isothiocyanate-tagged rabbit polyclonal antibody against DNMT1, 3A, -3B, or UBF ( panels a–t ). In a separate set, cells were stained with fluorescein isothiocyanate-tagged rabbit polyclonal antibody against UBF and TRITC-tagged mouse monoclonal antibody against DNMT1 ( panels u–y ). All five sets of cells were also stained with 4′,6-diamidino-2-phenylindole and visualized under a fluorescence microscope. B , all three DNMTs co-fractionated with nucleolin in the nucleolar fraction: nucleolar extract and nuclear extract (nucleolus and nucleoplasm) from HeLa cells (250 μ g) were subjected to Western blot analysis with antibodies against nucleolin, RNA polymerase II, DNMT1, -3A, and -3B. C , DNMT1, -3B, and -3A are associated with methylated rDNA promoter. Formaldehyde cross-linked chromatin was precleared and immunoprecipitated overnight with antisera specific for DNMT1, DNMT3A, DNMT3B, UBF, or preimmune sera. The immune complexes were precipitated by protein A/G beads, washed with different buffers (detailed under “Experimental Procedures”), eluted, and un-cross-linked. DNAs pulled down by different antibodies as well as input DNA were divided into three identical fractions that were either mock digested or digested with HpaII or MspI. An aliquot of each digestion product was subjected to real time PCR with primers specific for rDNA promoter. Association of different DNMTs and UBF with the rDNA promoter was analyzed using a standard curve generated by serial dilution of the undigested input DNA. Association with methylated promoter equals HpaII signal in ChIP DNA/HpaII signal in input (1:300 dilution). Association with unmethylated promoter corresponds to the signal in undigested minus signal in HpaII-digested ChIP DNA/Input signal in undigested minus HpaII-digested DNA (1:300 dilution). U and M indicate methylated and unmethylated rDNA promoters, respectively.
    Figure Legend Snippet: A , all three DNMTs localize in the nucleolus. HeLa cells were stained with TRITC-tagged mouse monoclonal antibody against nucleolin and with fluorescein isothiocyanate-tagged rabbit polyclonal antibody against DNMT1, 3A, -3B, or UBF ( panels a–t ). In a separate set, cells were stained with fluorescein isothiocyanate-tagged rabbit polyclonal antibody against UBF and TRITC-tagged mouse monoclonal antibody against DNMT1 ( panels u–y ). All five sets of cells were also stained with 4′,6-diamidino-2-phenylindole and visualized under a fluorescence microscope. B , all three DNMTs co-fractionated with nucleolin in the nucleolar fraction: nucleolar extract and nuclear extract (nucleolus and nucleoplasm) from HeLa cells (250 μ g) were subjected to Western blot analysis with antibodies against nucleolin, RNA polymerase II, DNMT1, -3A, and -3B. C , DNMT1, -3B, and -3A are associated with methylated rDNA promoter. Formaldehyde cross-linked chromatin was precleared and immunoprecipitated overnight with antisera specific for DNMT1, DNMT3A, DNMT3B, UBF, or preimmune sera. The immune complexes were precipitated by protein A/G beads, washed with different buffers (detailed under “Experimental Procedures”), eluted, and un-cross-linked. DNAs pulled down by different antibodies as well as input DNA were divided into three identical fractions that were either mock digested or digested with HpaII or MspI. An aliquot of each digestion product was subjected to real time PCR with primers specific for rDNA promoter. Association of different DNMTs and UBF with the rDNA promoter was analyzed using a standard curve generated by serial dilution of the undigested input DNA. Association with methylated promoter equals HpaII signal in ChIP DNA/HpaII signal in input (1:300 dilution). Association with unmethylated promoter corresponds to the signal in undigested minus signal in HpaII-digested ChIP DNA/Input signal in undigested minus HpaII-digested DNA (1:300 dilution). U and M indicate methylated and unmethylated rDNA promoters, respectively.

    Techniques Used: Staining, Fluorescence, Microscopy, Western Blot, Methylation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Generated, Serial Dilution, Chromatin Immunoprecipitation

    Catalytic domain of DNMT1 is necessary to inhibit unmethylated rDNA promoter that is de novo methylated by ectopic DNMT1 A , schematic diagram of different deletion/point mutants of DNMT1 used in transient transfection studies. B , HeLa cells were transfected with mock methylated pHrD-IRES-Luc and empty or expression vectors for different deletion mutants of DNMT1 (Δ CAT , catalytic domain deleted; CS , cysteine to serine mutation at PCQ motif; Δ NLS , NH 2 -terminal domain deleted including the nuclear localization signal). After 48 h firefly luciferase activity was measured in cell extracts. Results are represented as rDNA promoter activity upon overexpression of different DNMT1 mutants. One hundred percent activity represents empty vector-transfected pHrD-IRES-Luc activity. C , HeLa cells were cotransfected with mock-methylated pHrD-IRES-Luc and either empty vector or DNMT1 expression vector. The pHrD-IRES-Luc plasmid was isolated from the transfected cells 48 h post-transfection and digested with HpaII or MspI. The rDNA promoter was amplified from mock digested ( U ), HpaII ( H ), or MspI ( M ) digested plasmid using a vector-specific and a rDNA promoter-specific primer and resolved on 1% agarose gel.
    Figure Legend Snippet: Catalytic domain of DNMT1 is necessary to inhibit unmethylated rDNA promoter that is de novo methylated by ectopic DNMT1 A , schematic diagram of different deletion/point mutants of DNMT1 used in transient transfection studies. B , HeLa cells were transfected with mock methylated pHrD-IRES-Luc and empty or expression vectors for different deletion mutants of DNMT1 (Δ CAT , catalytic domain deleted; CS , cysteine to serine mutation at PCQ motif; Δ NLS , NH 2 -terminal domain deleted including the nuclear localization signal). After 48 h firefly luciferase activity was measured in cell extracts. Results are represented as rDNA promoter activity upon overexpression of different DNMT1 mutants. One hundred percent activity represents empty vector-transfected pHrD-IRES-Luc activity. C , HeLa cells were cotransfected with mock-methylated pHrD-IRES-Luc and either empty vector or DNMT1 expression vector. The pHrD-IRES-Luc plasmid was isolated from the transfected cells 48 h post-transfection and digested with HpaII or MspI. The rDNA promoter was amplified from mock digested ( U ), HpaII ( H ), or MspI ( M ) digested plasmid using a vector-specific and a rDNA promoter-specific primer and resolved on 1% agarose gel.

    Techniques Used: Methylation, Transfection, Expressing, Mutagenesis, Luciferase, Activity Assay, Over Expression, Plasmid Preparation, Isolation, Amplification, Agarose Gel Electrophoresis

    DNMT1 and -3B synergistically maintain rDNA promoter methylation A , whole cell extracts from HCT116 ( WT ), DNMT1 −/− ( MT1KO ), DNMT3B −/− ( 3BKO ), and DNMT1 −/− /DNMT3B −/− ( DKO ) cells were separated by SDS-PAGE and Western blot analysis was performed with specific antibodies. B , schematic presentation of the rDNA promoter region used as probe for Southern blot analysis. C , genomic DNA from the cells were either mock-digested ( U , Uncut), and digested with Hpall ( H ) or MspI ( M ). The digested products were separated on a 0.8% agarose gel and stained with ethidium bromide. D , DNA was transferred to nylon membrane and probed with a 32 P-labled rDNA promoter probe. E , rDNA, GAPDH, and albumin promoters were amplified from mock-digested ( U , Uncut), Hpall ( H ), or MspI ( M ) digested genomic DNA from W T HCT116, MT1KO, 3BKO, and DKO cells and resolved on 1.5% agarose gel. F , quantitative analysis of the PCR amplified rDNA products. G , total RNA isolated from the cells was resolved on a 1.0% agarose/formaldehyde gel, transferred to nylon membrane, and probed with the 91-bp pre-RNA probe. H , total RNA from the cells was treated with RNase-free DNase I, reverse transcribed, and subjected to real time PCR using 47 S rRNA-specific primers. Data presented was normalized to 18 S rRNA. RNA without reverse transcription did not generate PCR product.
    Figure Legend Snippet: DNMT1 and -3B synergistically maintain rDNA promoter methylation A , whole cell extracts from HCT116 ( WT ), DNMT1 −/− ( MT1KO ), DNMT3B −/− ( 3BKO ), and DNMT1 −/− /DNMT3B −/− ( DKO ) cells were separated by SDS-PAGE and Western blot analysis was performed with specific antibodies. B , schematic presentation of the rDNA promoter region used as probe for Southern blot analysis. C , genomic DNA from the cells were either mock-digested ( U , Uncut), and digested with Hpall ( H ) or MspI ( M ). The digested products were separated on a 0.8% agarose gel and stained with ethidium bromide. D , DNA was transferred to nylon membrane and probed with a 32 P-labled rDNA promoter probe. E , rDNA, GAPDH, and albumin promoters were amplified from mock-digested ( U , Uncut), Hpall ( H ), or MspI ( M ) digested genomic DNA from W T HCT116, MT1KO, 3BKO, and DKO cells and resolved on 1.5% agarose gel. F , quantitative analysis of the PCR amplified rDNA products. G , total RNA isolated from the cells was resolved on a 1.0% agarose/formaldehyde gel, transferred to nylon membrane, and probed with the 91-bp pre-RNA probe. H , total RNA from the cells was treated with RNase-free DNase I, reverse transcribed, and subjected to real time PCR using 47 S rRNA-specific primers. Data presented was normalized to 18 S rRNA. RNA without reverse transcription did not generate PCR product.

    Techniques Used: Methylation, SDS Page, Western Blot, Southern Blot, Agarose Gel Electrophoresis, Staining, Amplification, Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction

    4) Product Images from "PKC isoforms interact with and phosphorylate DNMT1"

    Article Title: PKC isoforms interact with and phosphorylate DNMT1

    Journal: BMC Biology

    doi: 10.1186/1741-7007-9-31

    DNMT1 and PKCζ colocalize in the nucleus of HeLa cells . HeLa cells are shown with ( A ), DsRed.DNMT1 (red) ( B ), GFP-phosphorylated-PKCζ (green) ( C ), DsRed.DNMT1 and GFP-phosphorylated-PKCζ (merged yellow) ( D ), nucleus (blue) ( E ), merge nucleus and DsRed.DNMT1 ( F ), merge nucleus and GFP-phosphorylated-PKCζ ( G ), merge nucleus, DsRed.DNMT1, and GFP-phosphorylated-PKCζ ( H ). The construct DsRed.DNMT1 was transfected in HeLa cells 48 hours before cells fixation and permeabilization. An anti-phosphorylated-PKCζ rabbit antibody was used in combination with an anti-rabbit antibody coupled with GFP to detect endogenous activated form of PKCζ.
    Figure Legend Snippet: DNMT1 and PKCζ colocalize in the nucleus of HeLa cells . HeLa cells are shown with ( A ), DsRed.DNMT1 (red) ( B ), GFP-phosphorylated-PKCζ (green) ( C ), DsRed.DNMT1 and GFP-phosphorylated-PKCζ (merged yellow) ( D ), nucleus (blue) ( E ), merge nucleus and DsRed.DNMT1 ( F ), merge nucleus and GFP-phosphorylated-PKCζ ( G ), merge nucleus, DsRed.DNMT1, and GFP-phosphorylated-PKCζ ( H ). The construct DsRed.DNMT1 was transfected in HeLa cells 48 hours before cells fixation and permeabilization. An anti-phosphorylated-PKCζ rabbit antibody was used in combination with an anti-rabbit antibody coupled with GFP to detect endogenous activated form of PKCζ.

    Techniques Used: Construct, Transfection

    Phosphorylation of DNMT1 by PKCζ reduces its methyltransferase activity . Quantitative measurements of S -adenosyl-l-( methyl - 3 H)methionine integration in a DNA matrix poly(dI-dC).poly(dI-dC) by 20 nM of recombinant DNMT1 in the presence 100 ng of recombinant PKCζ incubated with or without 50 μM of ATP for different times. Data are representative of three independent experiments. Bars, S.D.
    Figure Legend Snippet: Phosphorylation of DNMT1 by PKCζ reduces its methyltransferase activity . Quantitative measurements of S -adenosyl-l-( methyl - 3 H)methionine integration in a DNA matrix poly(dI-dC).poly(dI-dC) by 20 nM of recombinant DNMT1 in the presence 100 ng of recombinant PKCζ incubated with or without 50 μM of ATP for different times. Data are representative of three independent experiments. Bars, S.D.

    Techniques Used: Activity Assay, Recombinant, Incubation

    Decrease of DNA methylation in HEK-293 cells overexpressing DNMT1 and PKCζ . ( A ) Western blot analysis showing expression of PKCζ and DNMT1 in HEK-293 transfected cells used in the analysis of methylated DNA Ip-on-Chip described in Materials and methods. ( B ) Histograms representing the methylation status of 15 genes selected from active regions as measured by qPCR using DNA immunoprecipated with an antibody against 5-methylcytosine. Untr12 was used as a control for a negative region. TRPA1 was used as a positive control. Copy number values were normalized for primer efficiency by dividing by the values obtained using input DNA and the same primer pairs. Error bars represent standard deviations calculated from the triplicate determinations. *, P
    Figure Legend Snippet: Decrease of DNA methylation in HEK-293 cells overexpressing DNMT1 and PKCζ . ( A ) Western blot analysis showing expression of PKCζ and DNMT1 in HEK-293 transfected cells used in the analysis of methylated DNA Ip-on-Chip described in Materials and methods. ( B ) Histograms representing the methylation status of 15 genes selected from active regions as measured by qPCR using DNA immunoprecipated with an antibody against 5-methylcytosine. Untr12 was used as a control for a negative region. TRPA1 was used as a positive control. Copy number values were normalized for primer efficiency by dividing by the values obtained using input DNA and the same primer pairs. Error bars represent standard deviations calculated from the triplicate determinations. *, P

    Techniques Used: DNA Methylation Assay, Western Blot, Expressing, Transfection, Methylation, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Positive Control

    PKC isoforms preferentially phosphorylate DNMT1 N-terminal domain . ( A ) Diagram of DNMT1 showing the corresponding regions of the GST fusion DNMT1 fragments used for phosphorylation assays. Methylation DNA-dependent allosteric activation (MDDAAD), bromo domain (BD), and nuclear localization sequences (NLS) of DNMT1 are indicated. ( B ) Coomassie-stained gel representing GST fusion DNMT1 proteins used for phosphorylation assays. Positions of the fusion fragments are marked with an asterisk. ( C ) Phosphorylation of GST fusion DNMT1 fragments following incubation with 20 nM of activated recombinant PKCα, βI, βII, γ, δ or η using (γ- 32 P)ATP. Counts were obtained following subtraction of the negative control (GST alone). Data are representative of three independent experiments. ( D ) Phosphorylation of the GST fusion DNMT1 fragment 1 to 446 following incubation with 20 nM of activated recombinant PKCα, βI, βII, γ, δ, ε, η, μ or ζ using (γ- 32 P)ATP. Counts were obtained following subtraction of the negative control (GST alone). Data represent the average of three independent experiments that gave similar results. Bars, S.D.
    Figure Legend Snippet: PKC isoforms preferentially phosphorylate DNMT1 N-terminal domain . ( A ) Diagram of DNMT1 showing the corresponding regions of the GST fusion DNMT1 fragments used for phosphorylation assays. Methylation DNA-dependent allosteric activation (MDDAAD), bromo domain (BD), and nuclear localization sequences (NLS) of DNMT1 are indicated. ( B ) Coomassie-stained gel representing GST fusion DNMT1 proteins used for phosphorylation assays. Positions of the fusion fragments are marked with an asterisk. ( C ) Phosphorylation of GST fusion DNMT1 fragments following incubation with 20 nM of activated recombinant PKCα, βI, βII, γ, δ or η using (γ- 32 P)ATP. Counts were obtained following subtraction of the negative control (GST alone). Data are representative of three independent experiments. ( D ) Phosphorylation of the GST fusion DNMT1 fragment 1 to 446 following incubation with 20 nM of activated recombinant PKCα, βI, βII, γ, δ, ε, η, μ or ζ using (γ- 32 P)ATP. Counts were obtained following subtraction of the negative control (GST alone). Data represent the average of three independent experiments that gave similar results. Bars, S.D.

    Techniques Used: Methylation, Activation Assay, Staining, Incubation, Recombinant, Negative Control

    PKCε does not phosphorylate individual domains of DNMT1 . Incorporation of (γ- 32 P)ATP by GST fusion DNMT1 fragments following incubation with 20 nM of activated recombinant PKCζ, PKCμ or PKCε. Counts were obtained following subtraction of the negative control (GST alone). Data are representative of three independent experiments.
    Figure Legend Snippet: PKCε does not phosphorylate individual domains of DNMT1 . Incorporation of (γ- 32 P)ATP by GST fusion DNMT1 fragments following incubation with 20 nM of activated recombinant PKCζ, PKCμ or PKCε. Counts were obtained following subtraction of the negative control (GST alone). Data are representative of three independent experiments.

    Techniques Used: Incubation, Recombinant, Negative Control

    PKCζ interacts with and phosphorylates DNMT1 fragments . ( A ) Binding of PKCζ to GST fusion DNMT1 fragments using the pull-down procedure described in Materials and methods. Input, 10 ng of recombinant PKCζ. ( B ) Ponceau-stained transferred proteins from pull-down experiments. Positions of the fusion proteins are marked with an asterisk. ( C ) Phosphorylation of GST fusion DNMT1 fragments bound and ( D ) unbound to beads following incubation with 20 nM of activated recombinant PKCζ using (γ- 32 P)ATP. Counts were obtained following subtraction of the negative control (GST alone). Data are representative of three independent experiments. Bars, S.D.
    Figure Legend Snippet: PKCζ interacts with and phosphorylates DNMT1 fragments . ( A ) Binding of PKCζ to GST fusion DNMT1 fragments using the pull-down procedure described in Materials and methods. Input, 10 ng of recombinant PKCζ. ( B ) Ponceau-stained transferred proteins from pull-down experiments. Positions of the fusion proteins are marked with an asterisk. ( C ) Phosphorylation of GST fusion DNMT1 fragments bound and ( D ) unbound to beads following incubation with 20 nM of activated recombinant PKCζ using (γ- 32 P)ATP. Counts were obtained following subtraction of the negative control (GST alone). Data are representative of three independent experiments. Bars, S.D.

    Techniques Used: Binding Assay, Recombinant, Staining, Incubation, Negative Control

    In vivo association between DNMT1 and PKCζ . (A) Co-immunoprecipitation of DNMT1 and PKCζ in nuclear extracts of HEK-293 cells. The cells were transfected with DNMT1 and PKCζ-c-myc or c-myc for 48 hours and c-myc proteins were purified with immobilized anti-c-myc beads. Protein complexes were resolved by SDS/PAGE and the presence of PKCζ was demonstrated using an anti-c-myc antibody; DNMT1 and actin were revealed, respectively, using an anti-DNMT1 and an anti-actin antibody. (B) Detection of endogenous PKCζ activity in DNMT1 immunoprecipitates. Nuclear proteins from HEK-293 cells were incubated with beads prebound to an isotopic IgG antibody or antibodies against DNMT1 or PKCζ for 4 hours. After several washes, protein-bead complexes were tested for kinase activity using (γ- 32 P)ATP and PKCζ specific substrate. Data are representative of three independent experiments. rec. PKCζ, recombinant PKCζ.
    Figure Legend Snippet: In vivo association between DNMT1 and PKCζ . (A) Co-immunoprecipitation of DNMT1 and PKCζ in nuclear extracts of HEK-293 cells. The cells were transfected with DNMT1 and PKCζ-c-myc or c-myc for 48 hours and c-myc proteins were purified with immobilized anti-c-myc beads. Protein complexes were resolved by SDS/PAGE and the presence of PKCζ was demonstrated using an anti-c-myc antibody; DNMT1 and actin were revealed, respectively, using an anti-DNMT1 and an anti-actin antibody. (B) Detection of endogenous PKCζ activity in DNMT1 immunoprecipitates. Nuclear proteins from HEK-293 cells were incubated with beads prebound to an isotopic IgG antibody or antibodies against DNMT1 or PKCζ for 4 hours. After several washes, protein-bead complexes were tested for kinase activity using (γ- 32 P)ATP and PKCζ specific substrate. Data are representative of three independent experiments. rec. PKCζ, recombinant PKCζ.

    Techniques Used: In Vivo, Immunoprecipitation, Transfection, Purification, SDS Page, Activity Assay, Incubation, Recombinant

    PKC isoforms phosphorylate human recombinant DNMT1 . ( A ) Quantitative measurements of phosphorylation of 5 nM of DNMT1 in the presence of (γ- 32 P)ATP for 30 minutes at 30°C with the indicated amounts of activated recombinant human PKCα, δ, ε, μ or ζ. DNMT1 phosphorylation was quantified as the ratio of PKC activity to negative control. Data represent the average of two representative independent experiments. Bars, S.D. ( B ) PKC activity of recombinant PKC isoforms against CREB, showing that all isoforms were active. 20 nM of each PKC and 1.5 μM of CREB peptides were used for the assay and were incubated in the presence of (γ- 32 P)ATP for 30 minutes at 30°C. Bars, S.D. ( C ) Autoradiography of a SDS-PAGE showing incorporation (γ- 32 P)ATP in recombinant human DNMT1 following incubation with different amounts of human PKCζ.
    Figure Legend Snippet: PKC isoforms phosphorylate human recombinant DNMT1 . ( A ) Quantitative measurements of phosphorylation of 5 nM of DNMT1 in the presence of (γ- 32 P)ATP for 30 minutes at 30°C with the indicated amounts of activated recombinant human PKCα, δ, ε, μ or ζ. DNMT1 phosphorylation was quantified as the ratio of PKC activity to negative control. Data represent the average of two representative independent experiments. Bars, S.D. ( B ) PKC activity of recombinant PKC isoforms against CREB, showing that all isoforms were active. 20 nM of each PKC and 1.5 μM of CREB peptides were used for the assay and were incubated in the presence of (γ- 32 P)ATP for 30 minutes at 30°C. Bars, S.D. ( C ) Autoradiography of a SDS-PAGE showing incorporation (γ- 32 P)ATP in recombinant human DNMT1 following incubation with different amounts of human PKCζ.

    Techniques Used: Recombinant, Activity Assay, Negative Control, Incubation, Autoradiography, SDS Page

    5) Product Images from "A nucleolin-DNMT1 regulatory axis in acute myeloid leukemogenesis"

    Article Title: A nucleolin-DNMT1 regulatory axis in acute myeloid leukemogenesis

    Journal: Oncotarget

    doi:

    NCL inactivation contributes to the restoration of p15 INK4B gene in leukemia cells A and B, qPCR analysis of p15 INK4B expression in MV4-11 or Kasumi-1 cells transfected with NCL siRNA (A) or treated with AS1411 (B) as well as the corresponding controls for 48 hours. C, Schematic representation of CpG sites in p15 INK4B promoter for Bisulfite sequencing. CpG locations are indicated as vertical bars in the promoter of p15 INK4B (top). Arrows indicate the bisulfite sequencing region (bottom). D and E, Bisulfite sequencing of p15 INK4B gene promoter in MV4-11 cells treated with NCL inhibitor (D) or transfected with NCL siRNA (E) for 48 hours. F and G, Bisulfite sequencing of p15 INK4B gene promoter in Kasumi-1 cells treated with NCL inhibitor (F) or transfected with NCL siRNA (G) for 48 hours. H, A working model illustrating the role of NCL-NFκB cascade in regulation of DNMT1-dependent DNA methylation program in leukemia cells. Note, solid lines represent pathways that are identified in the present study, dash lines indicate pathways that remain elusive. D-G, Open circles indicate unmethylated CpG sites, filled circles indicate methylated CpG sites. Results of 20 or 10 clones are presented.
    Figure Legend Snippet: NCL inactivation contributes to the restoration of p15 INK4B gene in leukemia cells A and B, qPCR analysis of p15 INK4B expression in MV4-11 or Kasumi-1 cells transfected with NCL siRNA (A) or treated with AS1411 (B) as well as the corresponding controls for 48 hours. C, Schematic representation of CpG sites in p15 INK4B promoter for Bisulfite sequencing. CpG locations are indicated as vertical bars in the promoter of p15 INK4B (top). Arrows indicate the bisulfite sequencing region (bottom). D and E, Bisulfite sequencing of p15 INK4B gene promoter in MV4-11 cells treated with NCL inhibitor (D) or transfected with NCL siRNA (E) for 48 hours. F and G, Bisulfite sequencing of p15 INK4B gene promoter in Kasumi-1 cells treated with NCL inhibitor (F) or transfected with NCL siRNA (G) for 48 hours. H, A working model illustrating the role of NCL-NFκB cascade in regulation of DNMT1-dependent DNA methylation program in leukemia cells. Note, solid lines represent pathways that are identified in the present study, dash lines indicate pathways that remain elusive. D-G, Open circles indicate unmethylated CpG sites, filled circles indicate methylated CpG sites. Results of 20 or 10 clones are presented.

    Techniques Used: Real-time Polymerase Chain Reaction, Expressing, Transfection, Methylation Sequencing, DNA Methylation Assay, Methylation, Clone Assay

    NCL positively regulates DNMT expression A and B, The analysis of GEO dataset GSE12417 [GPL570, AML, n = 79 (A); GPL96, AML, n = 163 (B)] showing the correlation between NCL and DNMT expression in leukemia patients. Correlation between NCL and DNMT1 or DNMT3A or DNMT3B was assessed by Spearman correlation. P
    Figure Legend Snippet: NCL positively regulates DNMT expression A and B, The analysis of GEO dataset GSE12417 [GPL570, AML, n = 79 (A); GPL96, AML, n = 163 (B)] showing the correlation between NCL and DNMT expression in leukemia patients. Correlation between NCL and DNMT1 or DNMT3A or DNMT3B was assessed by Spearman correlation. P

    Techniques Used: Expressing

    NCL modulates DNMT1 expression via NFκB pathway A, Kasumi-1 or MV4-11 cells were transfected with NCL siRNA or scramble for 48 hours and the cells were lysed for Western blot. * indicates the non-specific band. B, Kasumi-1 or MV4-11 cells were transfected with NCL expression or empty vector for 48 hours and subjected to Western blot. C, Schematic representation of luciferase constructs used for reporter assays. D, pGL3- DNMT1 constructs were co-transfected with NFκB expression or empty vector into 293T cells and luciferase activity was measured at 48 hours after transfection. E, 293T cells were co-transfected with pGL3- DNMT1 and NCL expression or empty vector. The luciferase activity was measured at 48 hour after transfection. F, MV4-11 or Kasumi-1 cells were transfected with NFκB siRNA or scramble for 48 hours and subjected to Western blot. G, Western blot to determine the alteration of NFκB targets in MV4-11 or Kasumi-1 cells transfected with NFκB expression or empty vector for 48 hours. H, Western blot for indicated target genes in MV4-11 or Kasumi-1 cells treated with NFκB inhibitor Bay 11-7082 for 12 hours. I, Western blot for the cleaved caspases in MV4-11 or Kasumi-1 cells exposed to Bay 11-7082 for 12 hours.
    Figure Legend Snippet: NCL modulates DNMT1 expression via NFκB pathway A, Kasumi-1 or MV4-11 cells were transfected with NCL siRNA or scramble for 48 hours and the cells were lysed for Western blot. * indicates the non-specific band. B, Kasumi-1 or MV4-11 cells were transfected with NCL expression or empty vector for 48 hours and subjected to Western blot. C, Schematic representation of luciferase constructs used for reporter assays. D, pGL3- DNMT1 constructs were co-transfected with NFκB expression or empty vector into 293T cells and luciferase activity was measured at 48 hours after transfection. E, 293T cells were co-transfected with pGL3- DNMT1 and NCL expression or empty vector. The luciferase activity was measured at 48 hour after transfection. F, MV4-11 or Kasumi-1 cells were transfected with NFκB siRNA or scramble for 48 hours and subjected to Western blot. G, Western blot to determine the alteration of NFκB targets in MV4-11 or Kasumi-1 cells transfected with NFκB expression or empty vector for 48 hours. H, Western blot for indicated target genes in MV4-11 or Kasumi-1 cells treated with NFκB inhibitor Bay 11-7082 for 12 hours. I, Western blot for the cleaved caspases in MV4-11 or Kasumi-1 cells exposed to Bay 11-7082 for 12 hours.

    Techniques Used: Expressing, Transfection, Western Blot, Plasmid Preparation, Luciferase, Construct, Activity Assay

    Pharmacological inhibition of NCL reduces DNA methylation and suppresses leukemia cell growth A, MV4-11 or Kasumi-1 cells were treated with AS1411 or CRO26 (1 or 3 μM, respectively) for 48 hours and the cells were lysed for Western blot. p-NCL, phosphorylated NCL. * indicates the non-specific band. B, Western blot to detect the change of NFκB and DNMT1 in MV4-11 or Kasumi-1 cells treated with AS1411 or CRO26 (1 or 3 μM, respectively) for 48 hours. p-NFκB, phosphorylated NFκB. * indicates the non-specific band. C, Genomic DNA was extracted from the above treated MV4-11 or Kasumi-1 cells and subjected to Dotblot using 5mC antibody. Lower: representative image of Dotblot; Upper: the quantification of dot intensities, mean ± SD. D, MV4-11 or Kasumi-1 cells were treated with 1 or 3 μM of AS1411 or CRO26, respectively, and subjected to colony-forming assays. The graph indicates the colony number from three independent experiments. P values were determined by a two-tailed Student's t-test, mean ± SD, *, P
    Figure Legend Snippet: Pharmacological inhibition of NCL reduces DNA methylation and suppresses leukemia cell growth A, MV4-11 or Kasumi-1 cells were treated with AS1411 or CRO26 (1 or 3 μM, respectively) for 48 hours and the cells were lysed for Western blot. p-NCL, phosphorylated NCL. * indicates the non-specific band. B, Western blot to detect the change of NFκB and DNMT1 in MV4-11 or Kasumi-1 cells treated with AS1411 or CRO26 (1 or 3 μM, respectively) for 48 hours. p-NFκB, phosphorylated NFκB. * indicates the non-specific band. C, Genomic DNA was extracted from the above treated MV4-11 or Kasumi-1 cells and subjected to Dotblot using 5mC antibody. Lower: representative image of Dotblot; Upper: the quantification of dot intensities, mean ± SD. D, MV4-11 or Kasumi-1 cells were treated with 1 or 3 μM of AS1411 or CRO26, respectively, and subjected to colony-forming assays. The graph indicates the colony number from three independent experiments. P values were determined by a two-tailed Student's t-test, mean ± SD, *, P

    Techniques Used: Inhibition, DNA Methylation Assay, Western Blot, Two Tailed Test

    6) Product Images from "Targeting the PD-L1/DNMT1 axis in acquired resistance to sorafenib in human hepatocellular carcinoma"

    Article Title: Targeting the PD-L1/DNMT1 axis in acquired resistance to sorafenib in human hepatocellular carcinoma

    Journal: Oncology Reports

    doi: 10.3892/or.2017.5722

    PD-L1 and DNMT1 are frequently overexpressed and positively correlated in HCC resistance to sorafenib. (A) GEO data were analyzed for PD-L1 and DNMT1 expression in sorafenib-sensitive mice versus sorafenib-resistant mice. (B) The analysis of GEO dataset GSE73571 showing the correlation between PD-L1 and DNMT1 expression in sorafenib-resistant mice, GSE73571. Correlation between PD-L1 and DNMT1 was assessed by Pearson correlation. P
    Figure Legend Snippet: PD-L1 and DNMT1 are frequently overexpressed and positively correlated in HCC resistance to sorafenib. (A) GEO data were analyzed for PD-L1 and DNMT1 expression in sorafenib-sensitive mice versus sorafenib-resistant mice. (B) The analysis of GEO dataset GSE73571 showing the correlation between PD-L1 and DNMT1 expression in sorafenib-resistant mice, GSE73571. Correlation between PD-L1 and DNMT1 was assessed by Pearson correlation. P

    Techniques Used: Expressing, Mouse Assay

    Inactivation of NFκB blocks PD-L1/Stat3/DNMT1 pathway in sorafenib-resistant HCC cells. (A) Western blotting for the protein expression of p-NFκB and total NFκB in HepG2 SR and HepG2 C cells. (B) Western blotting (left) and qPCR (right) for the indicated genes in HepG2 SR transfected with PD-L1 empty vector or shRNA. (C) HepG2 SR cells were transfected with NFκB shRNA or its control vector for 48 h and subjected to ChIP. The change of NFκB binding on PD-L1 promoter was assessed by qPCR. (D) qPCR for DNMT1, PDL1 and CDH1 levels in HepG2 SR cells treated with Bay 11 for 48 h. Data are mean ± SD, *P
    Figure Legend Snippet: Inactivation of NFκB blocks PD-L1/Stat3/DNMT1 pathway in sorafenib-resistant HCC cells. (A) Western blotting for the protein expression of p-NFκB and total NFκB in HepG2 SR and HepG2 C cells. (B) Western blotting (left) and qPCR (right) for the indicated genes in HepG2 SR transfected with PD-L1 empty vector or shRNA. (C) HepG2 SR cells were transfected with NFκB shRNA or its control vector for 48 h and subjected to ChIP. The change of NFκB binding on PD-L1 promoter was assessed by qPCR. (D) qPCR for DNMT1, PDL1 and CDH1 levels in HepG2 SR cells treated with Bay 11 for 48 h. Data are mean ± SD, *P

    Techniques Used: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Chromatin Immunoprecipitation, Binding Assay

    Genetic or pharmacological disruption of PD-L1 or DNMT1 sensitizes HCC resistance to sorafenib. (A) HepG2 SR and Huh7 SR cells were transfected with DNMT1 shRNA or empty vectors for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (B) HepG2 SR and Huh7 SR cells were transfected with PD-L1 shRNA or empty vectors for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (C) HepG2 SR and Huh7 SR cells were treated with decitabine for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (D) HepG2 SR and Huh7 SR cells were treated with Bay 11 for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (E) Resistant cells were treated with DMSO, decitabine, Bay 11, or decitabine plus Bay 11 for 6 h then subjected to colony-forming assay. Representative images of colony-forming assay (left) and the quantification of colonies (right). Data are mean ± SD, *P
    Figure Legend Snippet: Genetic or pharmacological disruption of PD-L1 or DNMT1 sensitizes HCC resistance to sorafenib. (A) HepG2 SR and Huh7 SR cells were transfected with DNMT1 shRNA or empty vectors for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (B) HepG2 SR and Huh7 SR cells were transfected with PD-L1 shRNA or empty vectors for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (C) HepG2 SR and Huh7 SR cells were treated with decitabine for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (D) HepG2 SR and Huh7 SR cells were treated with Bay 11 for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (E) Resistant cells were treated with DMSO, decitabine, Bay 11, or decitabine plus Bay 11 for 6 h then subjected to colony-forming assay. Representative images of colony-forming assay (left) and the quantification of colonies (right). Data are mean ± SD, *P

    Techniques Used: Transfection, shRNA, CCK-8 Assay

    PD-L1 induces DNMT1-dependent DNA hypomethylation and restores the expression of methylation-silenced CDH1. (A) GEO data were analyzed for CDH1 expression in sorafenib-sensitive mice versus sorafenib-resistant mice. (B) qPCR measuring the expression levels of CDH1 genes in HepG2 C and Huh7 C vs HepG2 SR and Huh7 SR cells. (C) qPCR for CDH1 expression in HepG2 SR and Huh7 SR cells transfected with empty or PD-L1 shRNA. (D) Bisulfite analysis for the change of DNA methylation in CDH1 promoter (transcription start site −251 to +139) in HepG2 SR cells transfected with empty or PD-L1 shRNA. Data are mean ± SD, *P
    Figure Legend Snippet: PD-L1 induces DNMT1-dependent DNA hypomethylation and restores the expression of methylation-silenced CDH1. (A) GEO data were analyzed for CDH1 expression in sorafenib-sensitive mice versus sorafenib-resistant mice. (B) qPCR measuring the expression levels of CDH1 genes in HepG2 C and Huh7 C vs HepG2 SR and Huh7 SR cells. (C) qPCR for CDH1 expression in HepG2 SR and Huh7 SR cells transfected with empty or PD-L1 shRNA. (D) Bisulfite analysis for the change of DNA methylation in CDH1 promoter (transcription start site −251 to +139) in HepG2 SR cells transfected with empty or PD-L1 shRNA. Data are mean ± SD, *P

    Techniques Used: Expressing, Methylation, Mouse Assay, Real-time Polymerase Chain Reaction, Transfection, shRNA, DNA Methylation Assay

    PD-L1 regulates DNMT1 through STAT3 signaling. (A) Western blotting (left) and qPCR (right) for DNMT1 and PD-L1 in HepG2 SR transfected with PD-L1 empty vector or shRNA. (B and C) Western blotting for the protein expression of p-STAT3 and total STAT3 in HepG2 SR and HepG2 C (B) or HepG2 SR transfected with PDL1 empty vector or shRNA (C). (D) HepG2 SR cells were transfected with STAT3 siRNA or its control vector for 48 h and subjected to ChIP. The change of STAT3 binding on DNMT1 promoter was assessed by qPCR. (E) 293T cells were transfected with pGL3-DNMT1 alone or plus STAT3 vectors for 48 h, followed by the measurement of luciferase activity. Data are mean ± SD, *P
    Figure Legend Snippet: PD-L1 regulates DNMT1 through STAT3 signaling. (A) Western blotting (left) and qPCR (right) for DNMT1 and PD-L1 in HepG2 SR transfected with PD-L1 empty vector or shRNA. (B and C) Western blotting for the protein expression of p-STAT3 and total STAT3 in HepG2 SR and HepG2 C (B) or HepG2 SR transfected with PDL1 empty vector or shRNA (C). (D) HepG2 SR cells were transfected with STAT3 siRNA or its control vector for 48 h and subjected to ChIP. The change of STAT3 binding on DNMT1 promoter was assessed by qPCR. (E) 293T cells were transfected with pGL3-DNMT1 alone or plus STAT3 vectors for 48 h, followed by the measurement of luciferase activity. Data are mean ± SD, *P

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Chromatin Immunoprecipitation, Binding Assay, Luciferase, Activity Assay

    7) Product Images from "The Reelin and GAD67 Promoters Are Activated by Epigenetic Drugs That Facilitate the Disruption of Local Repressor Complexes"

    Article Title: The Reelin and GAD67 Promoters Are Activated by Epigenetic Drugs That Facilitate the Disruption of Local Repressor Complexes

    Journal: Molecular Pharmacology

    doi: 10.1124/mol.108.051763

    HDAC inhibitors down-regulate DNMT1 expression and DNMT enzymatic activity. NT-2 cells were either not treated (Control) or treated with 5 μM MS-275 (48 h), 5 μM MS-IN (48 h), 0.3 μM TSA (24 h), or 5 mM VPA (24 h). After treatment,
    Figure Legend Snippet: HDAC inhibitors down-regulate DNMT1 expression and DNMT enzymatic activity. NT-2 cells were either not treated (Control) or treated with 5 μM MS-275 (48 h), 5 μM MS-IN (48 h), 0.3 μM TSA (24 h), or 5 mM VPA (24 h). After treatment,

    Techniques Used: Expressing, Activity Assay, Mass Spectrometry

    siRNA-mediated DNMT knock-down induces overexpression of the nontargeted DNMT proteins. siRNA pools (15 nM) targeting DNMT1, DNMT3A, and DNMT3B were transfected into NT-2 cells either individually or in different combinations. For all experiments,
    Figure Legend Snippet: siRNA-mediated DNMT knock-down induces overexpression of the nontargeted DNMT proteins. siRNA pools (15 nM) targeting DNMT1, DNMT3A, and DNMT3B were transfected into NT-2 cells either individually or in different combinations. For all experiments,

    Techniques Used: Over Expression, Transfection

    8) Product Images from "The DNA Methyltransferase DNMT1 and Tyrosine-Protein Kinase KIT Cooperatively Promote Resistance to 5-Aza-2′-deoxycytidine (Decitabine) and Midostaurin (PKC412) in Lung Cancer Cells *"

    Article Title: The DNA Methyltransferase DNMT1 and Tyrosine-Protein Kinase KIT Cooperatively Promote Resistance to 5-Aza-2′-deoxycytidine (Decitabine) and Midostaurin (PKC412) in Lung Cancer Cells *

    Journal: The Journal of Biological Chemistry

    doi: 10.1074/jbc.M114.633693

    DNA is remethylated in decitabine R . A , decitabine R or parental H1975 cells were recovered in drug-free medium for 96 h and subjected to Western blot ( left panel ) or qPCR ( right panel ). B , the measurement of DNMT1 expression by Western blot ( left panel
    Figure Legend Snippet: DNA is remethylated in decitabine R . A , decitabine R or parental H1975 cells were recovered in drug-free medium for 96 h and subjected to Western blot ( left panel ) or qPCR ( right panel ). B , the measurement of DNMT1 expression by Western blot ( left panel

    Techniques Used: Western Blot, Real-time Polymerase Chain Reaction, Expressing

    KIT and DNMT1 have reciprocal regulation in lung cancer cells. A , H1975 and A549 cells were transfected with DNMT1 siRNA or scramble for 48 h, and the whole cell lysates were subjected to Western blot. B , H1975 and A549 cells were transfected with DNMT1
    Figure Legend Snippet: KIT and DNMT1 have reciprocal regulation in lung cancer cells. A , H1975 and A549 cells were transfected with DNMT1 siRNA or scramble for 48 h, and the whole cell lysates were subjected to Western blot. B , H1975 and A549 cells were transfected with DNMT1

    Techniques Used: Transfection, Western Blot

    9) Product Images from "The de novo methylation activity of Dnmt3a is distinctly different than that of Dnmt1"

    Article Title: The de novo methylation activity of Dnmt3a is distinctly different than that of Dnmt1

    Journal: BMC Biochemistry

    doi: 10.1186/1471-2091-6-6

    Model for one of the de novo methylation processes carried out by Dnmt3a and Dnmt1. Dnmt3a initiates methylation on one DNA strand at its preferred CpG sites (filled circles). After DNA replication, Dnmt1 methylates the other strand (open circles) at these hemimethylated sites. The hemimethylated DNA may also stimulate Dnmt1 for further methylation at previous unmethylated CpG sites (open circles indicated with ?).
    Figure Legend Snippet: Model for one of the de novo methylation processes carried out by Dnmt3a and Dnmt1. Dnmt3a initiates methylation on one DNA strand at its preferred CpG sites (filled circles). After DNA replication, Dnmt1 methylates the other strand (open circles) at these hemimethylated sites. The hemimethylated DNA may also stimulate Dnmt1 for further methylation at previous unmethylated CpG sites (open circles indicated with ?).

    Techniques Used: Methylation

    In vitro methylation of oligonucleotides by DNA methyltransferases. A) Duplex 30-mer and 122-mer DNA substrates with hemimethylated top strand (HMTop), hemimethylated bottom strand (HMBot), both strands methylated (DM), and fully unmethylated (UM) were used in the in vitro 3 H-incorporation assay (counts per minute, CPM) to test the activity of purified Dnmt1. B) Duplex 30-mer and 122-mer DNA substrates with hemimethylated top strand (HMTop), hemimethylated bottom strand (HMBot), both strands methylated (DM), and fully unmethylated (UM) were used in the in vitro 3 H-incorporation assay (counts per minute, CPM) to test the activity of purified Dnmt3a. C) The single-stranded 122-mer DNA substrates, unmethylated top strand (SSTop), unmethylated bottom strand (SSBot), methylated top strand (SMTop), and methylated bottom strand (SMBot), were used to assay Dnmt1 activity in vitro. D) Single-stranded 122-mer DNA substrates, unmethylated top strand (SSTop), unmethylated bottom strand (SSBot), methylated top strand (SMTop), and methylated bottom strand (SMBot), were used to assay Dnmt3a activity in vitro.
    Figure Legend Snippet: In vitro methylation of oligonucleotides by DNA methyltransferases. A) Duplex 30-mer and 122-mer DNA substrates with hemimethylated top strand (HMTop), hemimethylated bottom strand (HMBot), both strands methylated (DM), and fully unmethylated (UM) were used in the in vitro 3 H-incorporation assay (counts per minute, CPM) to test the activity of purified Dnmt1. B) Duplex 30-mer and 122-mer DNA substrates with hemimethylated top strand (HMTop), hemimethylated bottom strand (HMBot), both strands methylated (DM), and fully unmethylated (UM) were used in the in vitro 3 H-incorporation assay (counts per minute, CPM) to test the activity of purified Dnmt3a. C) The single-stranded 122-mer DNA substrates, unmethylated top strand (SSTop), unmethylated bottom strand (SSBot), methylated top strand (SMTop), and methylated bottom strand (SMBot), were used to assay Dnmt1 activity in vitro. D) Single-stranded 122-mer DNA substrates, unmethylated top strand (SSTop), unmethylated bottom strand (SSBot), methylated top strand (SMTop), and methylated bottom strand (SMBot), were used to assay Dnmt3a activity in vitro.

    Techniques Used: In Vitro, Methylation, Activity Assay, Purification

    10) Product Images from "Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription *"

    Article Title: Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription *

    Journal: The Journal of biological chemistry

    doi: 10.1074/jbc.M601155200

    Ectopic DNMT1 and DNMT3B, but not DNMT3A, inhibit rDNA promoter activity irrespective of its methylation status A , HeLa cells were transiently transfected with DNMT1, DNMT3A, or DNMT3B expression vectors. Whole cell extracts from these cells were subjected to Western blot analysis with anti-FLAG M2 antibody. B , HeLa cells were transfected with either mock methylated pHrD-IRES-Luc ( Unmethylated ) or M.HhaI-methylated pHrD-IRES-Luc ( Methylated ) along with 4 μ g of the empty vector or pcDNMT1, pcDNMT3A, or pcDNMT3B. After 48 h firefly luciferase activity was measured in the cell extracts. Results are represented as rDNA promoter activity upon overexpression of different DNMTs with empty vector-transfected pHrD-IRES-Luc activity as 100.
    Figure Legend Snippet: Ectopic DNMT1 and DNMT3B, but not DNMT3A, inhibit rDNA promoter activity irrespective of its methylation status A , HeLa cells were transiently transfected with DNMT1, DNMT3A, or DNMT3B expression vectors. Whole cell extracts from these cells were subjected to Western blot analysis with anti-FLAG M2 antibody. B , HeLa cells were transfected with either mock methylated pHrD-IRES-Luc ( Unmethylated ) or M.HhaI-methylated pHrD-IRES-Luc ( Methylated ) along with 4 μ g of the empty vector or pcDNMT1, pcDNMT3A, or pcDNMT3B. After 48 h firefly luciferase activity was measured in the cell extracts. Results are represented as rDNA promoter activity upon overexpression of different DNMTs with empty vector-transfected pHrD-IRES-Luc activity as 100.

    Techniques Used: Activity Assay, Methylation, Transfection, Expressing, Western Blot, Plasmid Preparation, Luciferase, Over Expression

    A , all three DNMTs localize in the nucleolus. HeLa cells were stained with TRITC-tagged mouse monoclonal antibody against nucleolin and with fluorescein isothiocyanate-tagged rabbit polyclonal antibody against DNMT1, 3A, -3B, or UBF ( panels a–t ). In a separate set, cells were stained with fluorescein isothiocyanate-tagged rabbit polyclonal antibody against UBF and TRITC-tagged mouse monoclonal antibody against DNMT1 ( panels u–y ). All five sets of cells were also stained with 4′,6-diamidino-2-phenylindole and visualized under a fluorescence microscope. B , all three DNMTs co-fractionated with nucleolin in the nucleolar fraction: nucleolar extract and nuclear extract (nucleolus and nucleoplasm) from HeLa cells (250 μ g) were subjected to Western blot analysis with antibodies against nucleolin, RNA polymerase II, DNMT1, -3A, and -3B. C , DNMT1, -3B, and -3A are associated with methylated rDNA promoter. Formaldehyde cross-linked chromatin was precleared and immunoprecipitated overnight with antisera specific for DNMT1, DNMT3A, DNMT3B, UBF, or preimmune sera. The immune complexes were precipitated by protein A/G beads, washed with different buffers (detailed under “Experimental Procedures”), eluted, and un-cross-linked. DNAs pulled down by different antibodies as well as input DNA were divided into three identical fractions that were either mock digested or digested with HpaII or MspI. An aliquot of each digestion product was subjected to real time PCR with primers specific for rDNA promoter. Association of different DNMTs and UBF with the rDNA promoter was analyzed using a standard curve generated by serial dilution of the undigested input DNA. Association with methylated promoter equals HpaII signal in ChIP DNA/HpaII signal in input (1:300 dilution). Association with unmethylated promoter corresponds to the signal in undigested minus signal in HpaII-digested ChIP DNA/Input signal in undigested minus HpaII-digested DNA (1:300 dilution). U and M indicate methylated and unmethylated rDNA promoters, respectively.
    Figure Legend Snippet: A , all three DNMTs localize in the nucleolus. HeLa cells were stained with TRITC-tagged mouse monoclonal antibody against nucleolin and with fluorescein isothiocyanate-tagged rabbit polyclonal antibody against DNMT1, 3A, -3B, or UBF ( panels a–t ). In a separate set, cells were stained with fluorescein isothiocyanate-tagged rabbit polyclonal antibody against UBF and TRITC-tagged mouse monoclonal antibody against DNMT1 ( panels u–y ). All five sets of cells were also stained with 4′,6-diamidino-2-phenylindole and visualized under a fluorescence microscope. B , all three DNMTs co-fractionated with nucleolin in the nucleolar fraction: nucleolar extract and nuclear extract (nucleolus and nucleoplasm) from HeLa cells (250 μ g) were subjected to Western blot analysis with antibodies against nucleolin, RNA polymerase II, DNMT1, -3A, and -3B. C , DNMT1, -3B, and -3A are associated with methylated rDNA promoter. Formaldehyde cross-linked chromatin was precleared and immunoprecipitated overnight with antisera specific for DNMT1, DNMT3A, DNMT3B, UBF, or preimmune sera. The immune complexes were precipitated by protein A/G beads, washed with different buffers (detailed under “Experimental Procedures”), eluted, and un-cross-linked. DNAs pulled down by different antibodies as well as input DNA were divided into three identical fractions that were either mock digested or digested with HpaII or MspI. An aliquot of each digestion product was subjected to real time PCR with primers specific for rDNA promoter. Association of different DNMTs and UBF with the rDNA promoter was analyzed using a standard curve generated by serial dilution of the undigested input DNA. Association with methylated promoter equals HpaII signal in ChIP DNA/HpaII signal in input (1:300 dilution). Association with unmethylated promoter corresponds to the signal in undigested minus signal in HpaII-digested ChIP DNA/Input signal in undigested minus HpaII-digested DNA (1:300 dilution). U and M indicate methylated and unmethylated rDNA promoters, respectively.

    Techniques Used: Staining, Fluorescence, Microscopy, Western Blot, Methylation, Immunoprecipitation, Real-time Polymerase Chain Reaction, Generated, Serial Dilution, Chromatin Immunoprecipitation

    Catalytic domain of DNMT1 is necessary to inhibit unmethylated rDNA promoter that is de novo methylated by ectopic DNMT1 A , schematic diagram of different deletion/point mutants of DNMT1 used in transient transfection studies. B , HeLa cells were transfected with mock methylated pHrD-IRES-Luc and empty or expression vectors for different deletion mutants of DNMT1 (Δ CAT , catalytic domain deleted; CS , cysteine to serine mutation at PCQ motif; Δ NLS , NH 2 -terminal domain deleted including the nuclear localization signal). After 48 h firefly luciferase activity was measured in cell extracts. Results are represented as rDNA promoter activity upon overexpression of different DNMT1 mutants. One hundred percent activity represents empty vector-transfected pHrD-IRES-Luc activity. C , HeLa cells were cotransfected with mock-methylated pHrD-IRES-Luc and either empty vector or DNMT1 expression vector. The pHrD-IRES-Luc plasmid was isolated from the transfected cells 48 h post-transfection and digested with HpaII or MspI. The rDNA promoter was amplified from mock digested ( U ), HpaII ( H ), or MspI ( M ) digested plasmid using a vector-specific and a rDNA promoter-specific primer and resolved on 1% agarose gel.
    Figure Legend Snippet: Catalytic domain of DNMT1 is necessary to inhibit unmethylated rDNA promoter that is de novo methylated by ectopic DNMT1 A , schematic diagram of different deletion/point mutants of DNMT1 used in transient transfection studies. B , HeLa cells were transfected with mock methylated pHrD-IRES-Luc and empty or expression vectors for different deletion mutants of DNMT1 (Δ CAT , catalytic domain deleted; CS , cysteine to serine mutation at PCQ motif; Δ NLS , NH 2 -terminal domain deleted including the nuclear localization signal). After 48 h firefly luciferase activity was measured in cell extracts. Results are represented as rDNA promoter activity upon overexpression of different DNMT1 mutants. One hundred percent activity represents empty vector-transfected pHrD-IRES-Luc activity. C , HeLa cells were cotransfected with mock-methylated pHrD-IRES-Luc and either empty vector or DNMT1 expression vector. The pHrD-IRES-Luc plasmid was isolated from the transfected cells 48 h post-transfection and digested with HpaII or MspI. The rDNA promoter was amplified from mock digested ( U ), HpaII ( H ), or MspI ( M ) digested plasmid using a vector-specific and a rDNA promoter-specific primer and resolved on 1% agarose gel.

    Techniques Used: Methylation, Transfection, Expressing, Mutagenesis, Luciferase, Activity Assay, Over Expression, Plasmid Preparation, Isolation, Amplification, Agarose Gel Electrophoresis

    DNMT1 and -3B synergistically maintain rDNA promoter methylation A , whole cell extracts from HCT116 ( WT ), DNMT1 −/− ( MT1KO ), DNMT3B −/− ( 3BKO ), and DNMT1 −/− /DNMT3B −/− ( DKO ) cells were separated by SDS-PAGE and Western blot analysis was performed with specific antibodies. B , schematic presentation of the rDNA promoter region used as probe for Southern blot analysis. C , genomic DNA from the cells were either mock-digested ( U , Uncut), and digested with Hpall ( H ) or MspI ( M ). The digested products were separated on a 0.8% agarose gel and stained with ethidium bromide. D , DNA was transferred to nylon membrane and probed with a 32 P-labled rDNA promoter probe. E , rDNA, GAPDH, and albumin promoters were amplified from mock-digested ( U , Uncut), Hpall ( H ), or MspI ( M ) digested genomic DNA from W T HCT116, MT1KO, 3BKO, and DKO cells and resolved on 1.5% agarose gel. F , quantitative analysis of the PCR amplified rDNA products. G , total RNA isolated from the cells was resolved on a 1.0% agarose/formaldehyde gel, transferred to nylon membrane, and probed with the 91-bp pre-RNA probe. H , total RNA from the cells was treated with RNase-free DNase I, reverse transcribed, and subjected to real time PCR using 47 S rRNA-specific primers. Data presented was normalized to 18 S rRNA. RNA without reverse transcription did not generate PCR product.
    Figure Legend Snippet: DNMT1 and -3B synergistically maintain rDNA promoter methylation A , whole cell extracts from HCT116 ( WT ), DNMT1 −/− ( MT1KO ), DNMT3B −/− ( 3BKO ), and DNMT1 −/− /DNMT3B −/− ( DKO ) cells were separated by SDS-PAGE and Western blot analysis was performed with specific antibodies. B , schematic presentation of the rDNA promoter region used as probe for Southern blot analysis. C , genomic DNA from the cells were either mock-digested ( U , Uncut), and digested with Hpall ( H ) or MspI ( M ). The digested products were separated on a 0.8% agarose gel and stained with ethidium bromide. D , DNA was transferred to nylon membrane and probed with a 32 P-labled rDNA promoter probe. E , rDNA, GAPDH, and albumin promoters were amplified from mock-digested ( U , Uncut), Hpall ( H ), or MspI ( M ) digested genomic DNA from W T HCT116, MT1KO, 3BKO, and DKO cells and resolved on 1.5% agarose gel. F , quantitative analysis of the PCR amplified rDNA products. G , total RNA isolated from the cells was resolved on a 1.0% agarose/formaldehyde gel, transferred to nylon membrane, and probed with the 91-bp pre-RNA probe. H , total RNA from the cells was treated with RNase-free DNase I, reverse transcribed, and subjected to real time PCR using 47 S rRNA-specific primers. Data presented was normalized to 18 S rRNA. RNA without reverse transcription did not generate PCR product.

    Techniques Used: Methylation, SDS Page, Western Blot, Southern Blot, Agarose Gel Electrophoresis, Staining, Amplification, Polymerase Chain Reaction, Isolation, Real-time Polymerase Chain Reaction

    11) Product Images from "An upregulation of DNA-methyltransferase 1 and 3a expressed in telencephalic GABAergic neurons of schizophrenia patients is also detected in peripheral blood lymphocytes"

    Article Title: An upregulation of DNA-methyltransferase 1 and 3a expressed in telencephalic GABAergic neurons of schizophrenia patients is also detected in peripheral blood lymphocytes

    Journal: Schizophrenia research

    doi: 10.1016/j.schres.2009.03.020

    DNMT1 mRNA is overexpressed in the PBL of SZ patients
    Figure Legend Snippet: DNMT1 mRNA is overexpressed in the PBL of SZ patients

    Techniques Used:

    DNMT1 but not DNMT 3a mRNA is upregulated in BA 10 of SZP
    Figure Legend Snippet: DNMT1 but not DNMT 3a mRNA is upregulated in BA 10 of SZP

    Techniques Used:

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    Incubation:

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    Activity Assay:

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    In Situ Hybridization:

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    Article Snippet: NSUN2-S139A and NSUN2-S139E proteins generated by the TNT in vitro transcription/translation (rabbit reticulocyte) system (Promega, Madison, WI) were also used for the methyltransferase assay. .. Sss1 (New England Biolabs) and Dnmt1 (New England Biolabs) were used as positive and negative control, respectively.

    Article Title: MBD4 cooperates with DNMT1 to mediate methyl-DNA repression and protects mammalian cells from oxidative stress
    Article Snippet: The GST-MBD4 constructs, the GFP-MBD4 construct and pVIC1-MBD4 constructs were generated by PCR-based cloning procedures using the EcoRI and SalI sites of pGEX-5X-1 (GE healthcare) and peGFP-C2 (Clontech), and the NdeI and SmaI sites of pVIC1 (New England Biolabs). .. Antibodies used in this study are commercially available: MBD4 (Bethyl Laboratories #A301–634A); MBD4 (Sigma-Aldrich #M9817), DNMT1 (New England Biolabs #M0231L), Beta-Actin (Cell Signaling Technology #4970), HA (Cell Signaling Technology #2367), 6xHIS (Cell Signaling Technology #2366), CDKN1A/p21 (Cell Signaling Technology #2946), TP53 (DO-1, Santa Cruz), GAPDH (Abcam #ab9485), ORC2 (Santa Cruz #28742), cMYC (Santa Cruz #sc-40), phospho-H2AX (Upstate #05-636), H2AX (Bethyl Laboratories #A300–083A), MeCP2 (Abcam #ab3752), MBD1 (Abcam #ab2846) and MBD2 (Santa Cruz #sc-9397).

    Sequencing:

    Article Title: Role of Human Ribosomal RNA (rRNA) Promoter Methylation and of Methyl-CpG-binding Protein MBD2 in the Suppression of rRNA Gene Expression *
    Article Snippet: These hemimethylated plasmids were methylated at the complementary CpG base pair with AdoMet catalyzed by DNMT1 (New England Biolabs) at 37 °C. .. Methylation at the respective sites were confirmed by sequencing the bisulfite-converted plasmids with the primers 5′-TGTGTGGAGTTGGAGAGTG-3′ and 5′-TTGGGGTTGATTAGAGGG-3′ (for the positive strand) and 5′-CCCTCCTACAACCAAAAC-3′ and 5′-TGTGTGGTTGTGATGGTG-3′ (for the negative strand).

    Recombinant:

    Article Title: Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription *
    Article Snippet: .. Immunofluorescence analysis was performed of anti-nucleolin monoclonal antibody (C23), anti-UBF antibody (both from Santa Cruz), and antibodies raised against recombinant DNMT3A and -3B in our laboratory or DNMT1 (New England Biolabs) as described ( ). .. Nuclei were stained using 4′,6-diamidino-2-phenylindole in the mounting fluid.

    Immunofluorescence:

    Article Title: Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription *
    Article Snippet: .. Immunofluorescence analysis was performed of anti-nucleolin monoclonal antibody (C23), anti-UBF antibody (both from Santa Cruz), and antibodies raised against recombinant DNMT3A and -3B in our laboratory or DNMT1 (New England Biolabs) as described ( ). .. Nuclei were stained using 4′,6-diamidino-2-phenylindole in the mounting fluid.

    Nucleic Acid Electrophoresis:

    Article Title: Targeting the PD-L1/DNMT1 axis in acquired resistance to sorafenib in human hepatocellular carcinoma
    Article Snippet: Protein was resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and transferred onto PVDF membranes (Amersham, Piscataway, NJ, USA). .. The antibodies used were β-actin (Santa Cruz Biotechnology); DNMT1 (New England Biolabs, Ipswich, MA, USA), the anti-STAT3, anti-phospho-STAT3 (Tyr705), anti-NFκB and anti-phospho-NFκB, anti-PD-L1 (Cell Signaling Technology).

    Radioactivity:

    Article Title:
    Article Snippet: Sss1 (New England Biolabs) and Dnmt1 (New England Biolabs) were used as positive and negative control, respectively. .. After reaction at 37°C for 30 min (for His-NSUN2) or 5 min (for TNT-produced NSUN2), unincorporated labeled compound was removed by using MicroSpin G25 columns (GE Healthcare), and incorporated radioactivity was measured by liquid scintillation counting.

    Article Title: The de novo methylation activity of Dnmt3a is distinctly different than that of Dnmt1
    Article Snippet: The same assay was carried out for Dnmt1 (New England Biolabs) in a 25 ul reaction with 1.1 uM 3 H-AdoMet in the manufacturer's recommended buffer using 2.5 units of the enzyme. .. The radioactivity retained on the air-dried DE-81 filters was measured by scintillation counting (Packard Tri Carb 2100TR) with 2 ml of scintillation fluid.

    Fluorescence:

    Article Title: An upregulation of DNA-methyltransferase 1 and 3a expressed in telencephalic GABAergic neurons of schizophrenia patients is also detected in peripheral blood lymphocytes
    Article Snippet: Paragraph title: 2.6. Double in situ hybridization- immunohistochemistry and confocal fluorescence microscopy ... Using 16-μm sections, after the completion of DNMT1 or DNMT3a mRNA in situ hybridization, these sections were processed for immunohistochemistry using either antibodies directed against GAD65/67 (Chemicon, Temecula, CA, 1:2000) or DNMT1 (New England Biolabs, 1:500) ( , ).

    Methylation:

    Article Title: Role of Human Ribosomal RNA (rRNA) Promoter Methylation and of Methyl-CpG-binding Protein MBD2 in the Suppression of rRNA Gene Expression *
    Article Snippet: .. These hemimethylated plasmids were methylated at the complementary CpG base pair with AdoMet catalyzed by DNMT1 (New England Biolabs) at 37 °C. .. Methylated (with AdoMet) or mock-methylated (without AdoMet), ligated plasmids were resolved by agarose gel electrophoresis and purified using gel extraction kit (Qiagen), and concentration was measured at 260 nm.

    Article Title: The de novo methylation activity of Dnmt3a is distinctly different than that of Dnmt1
    Article Snippet: In vitro methylation was carried out for Dnmt3a and the Dnmt3a mutant in a 20 ul reaction with 314 fmol of enzyme in 10 mM TrisHCl (pH 8.0), 1 mM EDTA, 1 mM DTT, and 1.1 uM 3 H-AdoMet (New England Nuclear, 14.7 Ci/mmol) at 37°C overnight (16 hours). .. The same assay was carried out for Dnmt1 (New England Biolabs) in a 25 ul reaction with 1.1 uM 3 H-AdoMet in the manufacturer's recommended buffer using 2.5 units of the enzyme.

    Mutagenesis:

    Article Title: The de novo methylation activity of Dnmt3a is distinctly different than that of Dnmt1
    Article Snippet: In vitro methylation was carried out for Dnmt3a and the Dnmt3a mutant in a 20 ul reaction with 314 fmol of enzyme in 10 mM TrisHCl (pH 8.0), 1 mM EDTA, 1 mM DTT, and 1.1 uM 3 H-AdoMet (New England Nuclear, 14.7 Ci/mmol) at 37°C overnight (16 hours). .. The same assay was carried out for Dnmt1 (New England Biolabs) in a 25 ul reaction with 1.1 uM 3 H-AdoMet in the manufacturer's recommended buffer using 2.5 units of the enzyme.

    Immunohistochemistry:

    Article Title: An upregulation of DNA-methyltransferase 1 and 3a expressed in telencephalic GABAergic neurons of schizophrenia patients is also detected in peripheral blood lymphocytes
    Article Snippet: .. Using 16-μm sections, after the completion of DNMT1 or DNMT3a mRNA in situ hybridization, these sections were processed for immunohistochemistry using either antibodies directed against GAD65/67 (Chemicon, Temecula, CA, 1:2000) or DNMT1 (New England Biolabs, 1:500) ( , ). .. Cy-5-conjugated goat anti-rabbit IgG (Amersham Biosciences, Piscataway, NJ, 1:1000) was used to label the antibodies that reacted with DNMT1 or GAD65/67 .

    Microscopy:

    Article Title: An upregulation of DNA-methyltransferase 1 and 3a expressed in telencephalic GABAergic neurons of schizophrenia patients is also detected in peripheral blood lymphocytes
    Article Snippet: Paragraph title: 2.6. Double in situ hybridization- immunohistochemistry and confocal fluorescence microscopy ... Using 16-μm sections, after the completion of DNMT1 or DNMT3a mRNA in situ hybridization, these sections were processed for immunohistochemistry using either antibodies directed against GAD65/67 (Chemicon, Temecula, CA, 1:2000) or DNMT1 (New England Biolabs, 1:500) ( , ).

    Purification:

    Article Title: Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription *
    Article Snippet: For chromatin immunoprecipitation (ChIP) analysis, antisera against UBF (Santa Cruz), DNMT1 (New England Biolabs), DNMT3A and -3B raised in our laboratory, were used ( ). .. Immunoprecipitated DNA-protein complex was eluted, un-cross-linked, treated with RNase A and proteinase K, and purified as described ( ).

    Article Title: Role of Human Ribosomal RNA (rRNA) Promoter Methylation and of Methyl-CpG-binding Protein MBD2 in the Suppression of rRNA Gene Expression *
    Article Snippet: These hemimethylated plasmids were methylated at the complementary CpG base pair with AdoMet catalyzed by DNMT1 (New England Biolabs) at 37 °C. .. Methylated (with AdoMet) or mock-methylated (without AdoMet), ligated plasmids were resolved by agarose gel electrophoresis and purified using gel extraction kit (Qiagen), and concentration was measured at 260 nm.

    Article Title:
    Article Snippet: Sss1 (New England Biolabs) and Dnmt1 (New England Biolabs) were used as positive and negative control, respectively. .. In methyltransferase assay for hemimethylated DNA, hemimethylated mimic substrate poly(dI:dC) (Roche Diagnostics, Mannheim, Germany) (0.2 pmol) or tRNA (purified from E. coli ; Sigma-Aldrich) (4 nmol) was reacted with the kinase reacted His-NSUN2 (0.5 μM) or TNT-produced NSUN2 (0.5 μM) in Dnmt1 reaction buffer (New England Biolabs) containing 6.5 μM S -adenosyl-L-[ methyl -3 H]methionine (specific activity 555 GBq/mmol; GE Healthcare) and RNasein (40 U/μl; Promega).

    Article Title: The de novo methylation activity of Dnmt3a is distinctly different than that of Dnmt1
    Article Snippet: The GST-Dnmt3a (GST-3a) and GST-Dnmt3a mutant (GST-3aMut) fusion proteins were expressed in 293T cells and purified using glutathione-agarose beads as described previously [ ]. .. The same assay was carried out for Dnmt1 (New England Biolabs) in a 25 ul reaction with 1.1 uM 3 H-AdoMet in the manufacturer's recommended buffer using 2.5 units of the enzyme.

    Polymerase Chain Reaction:

    Article Title: MBD4 cooperates with DNMT1 to mediate methyl-DNA repression and protects mammalian cells from oxidative stress
    Article Snippet: All PCR amplifications were performed using Phusion DNA polymerase (Finnzyme). .. Antibodies used in this study are commercially available: MBD4 (Bethyl Laboratories #A301–634A); MBD4 (Sigma-Aldrich #M9817), DNMT1 (New England Biolabs #M0231L), Beta-Actin (Cell Signaling Technology #4970), HA (Cell Signaling Technology #2367), 6xHIS (Cell Signaling Technology #2366), CDKN1A/p21 (Cell Signaling Technology #2946), TP53 (DO-1, Santa Cruz), GAPDH (Abcam #ab9485), ORC2 (Santa Cruz #28742), cMYC (Santa Cruz #sc-40), phospho-H2AX (Upstate #05-636), H2AX (Bethyl Laboratories #A300–083A), MeCP2 (Abcam #ab3752), MBD1 (Abcam #ab2846) and MBD2 (Santa Cruz #sc-9397).

    Immunoprecipitation:

    Article Title: Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription *
    Article Snippet: For chromatin immunoprecipitation (ChIP) analysis, antisera against UBF (Santa Cruz), DNMT1 (New England Biolabs), DNMT3A and -3B raised in our laboratory, were used ( ). .. Immunoprecipitated DNA-protein complex was eluted, un-cross-linked, treated with RNase A and proteinase K, and purified as described ( ).

    Article Title: The DNA Methyltransferase DNMT1 and Tyrosine-Protein Kinase KIT Cooperatively Promote Resistance to 5-Aza-2′-deoxycytidine (Decitabine) and Midostaurin (PKC412) in Lung Cancer Cells *
    Article Snippet: Paragraph title: Immunoprecipitation and Western Blot ... The antibodies are: Sp1 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA); KIT, phospho-KIT (Tyr-719), AKT, phospho-AKT (Ser-473), STAT3, phospho-STAT3, STAT5, phospho-STAT5, and CDH1 (Cell Signaling Technology, Danvers, MA); DNMT1 (New England Biolabs, Ipswich, MA); and phosphotyrosine 4G10 (anti-4G10) (Millipore, Billerica, MA).

    Quantitative RT-PCR:

    Article Title: The Reelin and GAD67 Promoters Are Activated by Epigenetic Drugs That Facilitate the Disruption of Local Repressor Complexes
    Article Snippet: Primary antibodies used for Western blot analysis were as follows: DNMT1 (1:1000 dilution; New England Biolabs, Ipswich, MA), DNMT3A (1:400 dilution; a generous gift from Dr. Shoji Tajima, Osaka University, Osaka, Japan), DNMT3B (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and MeCP2 antibody (1:500 dilution; Millipore, Billerica, MA). .. Quantitative RT-PCR Analysis.

    Staining:

    Article Title: Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription *
    Article Snippet: Immunofluorescence analysis was performed of anti-nucleolin monoclonal antibody (C23), anti-UBF antibody (both from Santa Cruz), and antibodies raised against recombinant DNMT3A and -3B in our laboratory or DNMT1 (New England Biolabs) as described ( ). .. Nuclei were stained using 4′,6-diamidino-2-phenylindole in the mounting fluid.

    Chromatin Immunoprecipitation:

    Article Title: Role of DNA Methyltransferases in Regulation of Human Ribosomal RNA Gene Transcription *
    Article Snippet: .. For chromatin immunoprecipitation (ChIP) analysis, antisera against UBF (Santa Cruz), DNMT1 (New England Biolabs), DNMT3A and -3B raised in our laboratory, were used ( ). .. The chromatin was first pre-cleared with pre-immune sera coupled to protein A/G beads followed by overnight incubation with preimmune or immune sera.

    Article Title: The Reelin and GAD67 Promoters Are Activated by Epigenetic Drugs That Facilitate the Disruption of Local Repressor Complexes
    Article Snippet: Primary antibodies used for Western blot analysis were as follows: DNMT1 (1:1000 dilution; New England Biolabs, Ipswich, MA), DNMT3A (1:400 dilution; a generous gift from Dr. Shoji Tajima, Osaka University, Osaka, Japan), DNMT3B (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and MeCP2 antibody (1:500 dilution; Millipore, Billerica, MA). .. In addition, chromatin immunoprecipitation (ChIP) grade anti-DNMT1 monoclonal antibody (Imgenex, San Diego, CA), anti-DNMT3B and anti-MeCP2 polyclonal antibodies (Abcam, Cambridge, MA), anti-acetyl-histone H3 polyclonal antibody (Millipore), and DNMT3A (Santa Cruz Biotechnology) polyclonal antibody were used for ChIP assays.

    SDS Page:

    Article Title: The DNA Methyltransferase DNMT1 and Tyrosine-Protein Kinase KIT Cooperatively Promote Resistance to 5-Aza-2′-deoxycytidine (Decitabine) and Midostaurin (PKC412) in Lung Cancer Cells *
    Article Snippet: The immunoprecipitates or the whole cellular lysates were resolved by SDS-PAGE and transferred onto PVDF membranes (Amersham Biosciences) for Western blot ( , ). .. The antibodies are: Sp1 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA); KIT, phospho-KIT (Tyr-719), AKT, phospho-AKT (Ser-473), STAT3, phospho-STAT3, STAT5, phospho-STAT5, and CDH1 (Cell Signaling Technology, Danvers, MA); DNMT1 (New England Biolabs, Ipswich, MA); and phosphotyrosine 4G10 (anti-4G10) (Millipore, Billerica, MA).

    Article Title:
    Article Snippet: [35 S]methionine (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom) incorporation followed by SDS-PAGE and fluorography confirmed that a protein of the expected size was translated, and the NSUN2 protein amount was estimated by immunoblot. .. Sss1 (New England Biolabs) and Dnmt1 (New England Biolabs) were used as positive and negative control, respectively.

    Plasmid Preparation:

    Article Title: Role of Human Ribosomal RNA (rRNA) Promoter Methylation and of Methyl-CpG-binding Protein MBD2 in the Suppression of rRNA Gene Expression *
    Article Snippet: In a typical reaction, positive strand HrD-Luc plasmid (∼0.05 pmol) was annealed to 1.25 pmol of phosphorylated oligonucleotides (with a specific CpG either methylated denoted as “M” oligonucleotide or unmethylated control, depicted as “C” oligonucleotide) spanning different regions of the rRNA promoter or external transcribed spacer (external transcribed spacer region 1; ETS1). .. These hemimethylated plasmids were methylated at the complementary CpG base pair with AdoMet catalyzed by DNMT1 (New England Biolabs) at 37 °C.

    Negative Control:

    Article Title:
    Article Snippet: .. Sss1 (New England Biolabs) and Dnmt1 (New England Biolabs) were used as positive and negative control, respectively. .. After reaction at 37°C for 1 h, the reacted lambda DNA was digested with BstU I (New England Biolabs) and analyzed by agarose gel electrophoresis.

    Agarose Gel Electrophoresis:

    Article Title: Role of Human Ribosomal RNA (rRNA) Promoter Methylation and of Methyl-CpG-binding Protein MBD2 in the Suppression of rRNA Gene Expression *
    Article Snippet: These hemimethylated plasmids were methylated at the complementary CpG base pair with AdoMet catalyzed by DNMT1 (New England Biolabs) at 37 °C. .. Methylated (with AdoMet) or mock-methylated (without AdoMet), ligated plasmids were resolved by agarose gel electrophoresis and purified using gel extraction kit (Qiagen), and concentration was measured at 260 nm.

    Article Title:
    Article Snippet: Sss1 (New England Biolabs) and Dnmt1 (New England Biolabs) were used as positive and negative control, respectively. .. After reaction at 37°C for 1 h, the reacted lambda DNA was digested with BstU I (New England Biolabs) and analyzed by agarose gel electrophoresis.

    In Vitro:

    Article Title:
    Article Snippet: NSUN2-S139A and NSUN2-S139E proteins generated by the TNT in vitro transcription/translation (rabbit reticulocyte) system (Promega, Madison, WI) were also used for the methyltransferase assay. .. Sss1 (New England Biolabs) and Dnmt1 (New England Biolabs) were used as positive and negative control, respectively.

    Article Title: The de novo methylation activity of Dnmt3a is distinctly different than that of Dnmt1
    Article Snippet: Paragraph title: Enzymes and in vitro 3 H-incorporation assay ... The same assay was carried out for Dnmt1 (New England Biolabs) in a 25 ul reaction with 1.1 uM 3 H-AdoMet in the manufacturer's recommended buffer using 2.5 units of the enzyme.

    DNA Methylation Assay:

    Article Title: PKC isoforms interact with and phosphorylate DNMT1
    Article Snippet: Paragraph title: DNA methylation assay ... Briefly, 20 nM of DNMT1 (New England Biolabs) and 100 ng of PKCζ were incubated with or without 50 μM of ATP in the presence of 5 μCi of S -adenosyl-l-(methyl -3 H)methionine (AdoMet) and 50 ng of poly(dI-dC)·poly(dI-dC) in methyltransferase buffer (50 mM Tris-HCL, pH 7.8, 1 mM Na2 EDTA, pH 8.0, 1 mM DTT, 7 μg/ml phenylmethylsulfonyl fluoride, 5% glycerol) supplemented with 5 μg of phosphatidylserine and 5 mM MgCl2 to allow PKCζ activity.

    Concentration Assay:

    Article Title: UHRF1 binds G9a and participates in p21 transcriptional regulation in mammalian cells
    Article Snippet: .. For siRNA transfections, hUHRF1 (Invitrogen), G9a (New England Biolabs), DNMT1 (New England Biolabs) and control Litmus (New England Biolabs) siRNAs were transfected into HeLa cells twice to a final concentration of 20–100 nM for 4 days using RNAiFECT reagent (Qiagen). .. Cell synchronization at G1/S was performed essentially following the double thymidine block protocol ( ) except that aphidicolin (3 μg/ml) instead of thymidine (2 mM) was used for the first block.

    Article Title: Role of Human Ribosomal RNA (rRNA) Promoter Methylation and of Methyl-CpG-binding Protein MBD2 in the Suppression of rRNA Gene Expression *
    Article Snippet: The circularized plasmids were pooled, precipitated, and ligated overnight at 16 °C with a high concentration (10 units/ μ l) ligase (Invitrogen) in 10 μ l of ligation mixture. .. These hemimethylated plasmids were methylated at the complementary CpG base pair with AdoMet catalyzed by DNMT1 (New England Biolabs) at 37 °C.

    Article Title: The Reelin and GAD67 Promoters Are Activated by Epigenetic Drugs That Facilitate the Disruption of Local Repressor Complexes
    Article Snippet: MS-275 and its inactive 3′-aminophenylbenzamide derivative (MS-IN) ( ) were dissolved in 100% dimethyl sulfoxide (DMSO) to a concentration of 10-2 M and stored at -20°C. .. Primary antibodies used for Western blot analysis were as follows: DNMT1 (1:1000 dilution; New England Biolabs, Ipswich, MA), DNMT3A (1:400 dilution; a generous gift from Dr. Shoji Tajima, Osaka University, Osaka, Japan), DNMT3B (1:100 dilution; Santa Cruz Biotechnology, Santa Cruz, CA), and MeCP2 antibody (1:500 dilution; Millipore, Billerica, MA).

    Construct:

    Article Title: MBD4 cooperates with DNMT1 to mediate methyl-DNA repression and protects mammalian cells from oxidative stress
    Article Snippet: The GST-MBD4 constructs, the GFP-MBD4 construct and pVIC1-MBD4 constructs were generated by PCR-based cloning procedures using the EcoRI and SalI sites of pGEX-5X-1 (GE healthcare) and peGFP-C2 (Clontech), and the NdeI and SmaI sites of pVIC1 (New England Biolabs). .. Antibodies used in this study are commercially available: MBD4 (Bethyl Laboratories #A301–634A); MBD4 (Sigma-Aldrich #M9817), DNMT1 (New England Biolabs #M0231L), Beta-Actin (Cell Signaling Technology #4970), HA (Cell Signaling Technology #2367), 6xHIS (Cell Signaling Technology #2366), CDKN1A/p21 (Cell Signaling Technology #2946), TP53 (DO-1, Santa Cruz), GAPDH (Abcam #ab9485), ORC2 (Santa Cruz #28742), cMYC (Santa Cruz #sc-40), phospho-H2AX (Upstate #05-636), H2AX (Bethyl Laboratories #A300–083A), MeCP2 (Abcam #ab3752), MBD1 (Abcam #ab2846) and MBD2 (Santa Cruz #sc-9397).

    Lysis:

    Article Title: The DNA Methyltransferase DNMT1 and Tyrosine-Protein Kinase KIT Cooperatively Promote Resistance to 5-Aza-2′-deoxycytidine (Decitabine) and Midostaurin (PKC412) in Lung Cancer Cells *
    Article Snippet: After the various treatments, the whole cellular lysates were prepared in 1× cell lysis buffer ( , ). .. The antibodies are: Sp1 and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA); KIT, phospho-KIT (Tyr-719), AKT, phospho-AKT (Ser-473), STAT3, phospho-STAT3, STAT5, phospho-STAT5, and CDH1 (Cell Signaling Technology, Danvers, MA); DNMT1 (New England Biolabs, Ipswich, MA); and phosphotyrosine 4G10 (anti-4G10) (Millipore, Billerica, MA).

    Article Title: Targeting the PD-L1/DNMT1 axis in acquired resistance to sorafenib in human hepatocellular carcinoma
    Article Snippet: Western blotting After the various treatments, the whole cellular lysates were prepared by harvesting the cells in 1X cell lysis buffer [20 mM HEPES (pH 7.6), 150 mM NaCl and 0.1% NP40] supplemented with 1X phosphatase inhibitor Cocktail 2 and 3 (Sigma-Aldrich), 1 mM PMSF (Sigma-Aldrich) and 1X protease inhibitors (protease inhibitor cocktail set III, Calbiochem-Novabiochem, San Diego, CA, USA). .. The antibodies used were β-actin (Santa Cruz Biotechnology); DNMT1 (New England Biolabs, Ipswich, MA, USA), the anti-STAT3, anti-phospho-STAT3 (Tyr705), anti-NFκB and anti-phospho-NFκB, anti-PD-L1 (Cell Signaling Technology).

    Article Title: A nucleolin-DNMT1 regulatory axis in acute myeloid leukemogenesis
    Article Snippet: Briefly, upon different treatment, whole cell lysates were prepared by lysing the cells in 1× cell lysis buffer [20 mM HEPES (pH 7.6), 150 mM NaCl and 0.1% NP40] supplemented with 1 × Phosphatase Inhibitor Cocktail 2 and 3 (Sigma Aldrich), 1 mM PMSF (Sigma Aldrich) and 1 × protease inhibitors (protease inhibitor cocktail set III, Calbiochem-Novabiochem). .. The antibodies used were: -Actin and DNMT3A from Santa Cruz Biotechnology; phospho-NCL (Thr76/Thr84) from Biolengend; NCL, DNMT3B and NFκBp65 from Abcam; phospho-NFκBp65 (Ser536), cleaved Parp, cleaved caspase-3 and cleaved caspase-8 from Cell Signaling Technology; DNMT1 from New England Biolabs.

    Gel Extraction:

    Article Title: Role of Human Ribosomal RNA (rRNA) Promoter Methylation and of Methyl-CpG-binding Protein MBD2 in the Suppression of rRNA Gene Expression *
    Article Snippet: These hemimethylated plasmids were methylated at the complementary CpG base pair with AdoMet catalyzed by DNMT1 (New England Biolabs) at 37 °C. .. Methylated (with AdoMet) or mock-methylated (without AdoMet), ligated plasmids were resolved by agarose gel electrophoresis and purified using gel extraction kit (Qiagen), and concentration was measured at 260 nm.

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    New England Biolabs dnmt1
    PD-L1 and <t>DNMT1</t> are frequently overexpressed and positively correlated in HCC resistance to sorafenib. (A) GEO data were analyzed for PD-L1 and DNMT1 expression in sorafenib-sensitive mice versus sorafenib-resistant mice. (B) The analysis of GEO dataset GSE73571 showing the correlation between PD-L1 and DNMT1 expression in sorafenib-resistant mice, GSE73571. Correlation between PD-L1 and DNMT1 was assessed by Pearson correlation. P
    Dnmt1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 96/100, based on 4 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/dnmt1/product/New England Biolabs
    Average 96 stars, based on 4 article reviews
    Price from $9.99 to $1999.99
    dnmt1 - by Bioz Stars, 2020-02
    96/100 stars
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    99
    New England Biolabs human dnmt1
    <t>DNMT1</t> was degraded upon S G treatment and the degradation could be blocked by a proteasomal inhibitor
    Human Dnmt1, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 99/100, based on 5 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/human dnmt1/product/New England Biolabs
    Average 99 stars, based on 5 article reviews
    Price from $9.99 to $1999.99
    human dnmt1 - by Bioz Stars, 2020-02
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    PD-L1 and DNMT1 are frequently overexpressed and positively correlated in HCC resistance to sorafenib. (A) GEO data were analyzed for PD-L1 and DNMT1 expression in sorafenib-sensitive mice versus sorafenib-resistant mice. (B) The analysis of GEO dataset GSE73571 showing the correlation between PD-L1 and DNMT1 expression in sorafenib-resistant mice, GSE73571. Correlation between PD-L1 and DNMT1 was assessed by Pearson correlation. P

    Journal: Oncology Reports

    Article Title: Targeting the PD-L1/DNMT1 axis in acquired resistance to sorafenib in human hepatocellular carcinoma

    doi: 10.3892/or.2017.5722

    Figure Lengend Snippet: PD-L1 and DNMT1 are frequently overexpressed and positively correlated in HCC resistance to sorafenib. (A) GEO data were analyzed for PD-L1 and DNMT1 expression in sorafenib-sensitive mice versus sorafenib-resistant mice. (B) The analysis of GEO dataset GSE73571 showing the correlation between PD-L1 and DNMT1 expression in sorafenib-resistant mice, GSE73571. Correlation between PD-L1 and DNMT1 was assessed by Pearson correlation. P

    Article Snippet: DNMT3a and DNMT3b have mostly de novo DNA methylation activity, whereas DNMT1 plays a central role in preserving the patterns of DNA methylation through cell division ( ).

    Techniques: Expressing, Mouse Assay

    Inactivation of NFκB blocks PD-L1/Stat3/DNMT1 pathway in sorafenib-resistant HCC cells. (A) Western blotting for the protein expression of p-NFκB and total NFκB in HepG2 SR and HepG2 C cells. (B) Western blotting (left) and qPCR (right) for the indicated genes in HepG2 SR transfected with PD-L1 empty vector or shRNA. (C) HepG2 SR cells were transfected with NFκB shRNA or its control vector for 48 h and subjected to ChIP. The change of NFκB binding on PD-L1 promoter was assessed by qPCR. (D) qPCR for DNMT1, PDL1 and CDH1 levels in HepG2 SR cells treated with Bay 11 for 48 h. Data are mean ± SD, *P

    Journal: Oncology Reports

    Article Title: Targeting the PD-L1/DNMT1 axis in acquired resistance to sorafenib in human hepatocellular carcinoma

    doi: 10.3892/or.2017.5722

    Figure Lengend Snippet: Inactivation of NFκB blocks PD-L1/Stat3/DNMT1 pathway in sorafenib-resistant HCC cells. (A) Western blotting for the protein expression of p-NFκB and total NFκB in HepG2 SR and HepG2 C cells. (B) Western blotting (left) and qPCR (right) for the indicated genes in HepG2 SR transfected with PD-L1 empty vector or shRNA. (C) HepG2 SR cells were transfected with NFκB shRNA or its control vector for 48 h and subjected to ChIP. The change of NFκB binding on PD-L1 promoter was assessed by qPCR. (D) qPCR for DNMT1, PDL1 and CDH1 levels in HepG2 SR cells treated with Bay 11 for 48 h. Data are mean ± SD, *P

    Article Snippet: DNMT3a and DNMT3b have mostly de novo DNA methylation activity, whereas DNMT1 plays a central role in preserving the patterns of DNA methylation through cell division ( ).

    Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Chromatin Immunoprecipitation, Binding Assay

    Genetic or pharmacological disruption of PD-L1 or DNMT1 sensitizes HCC resistance to sorafenib. (A) HepG2 SR and Huh7 SR cells were transfected with DNMT1 shRNA or empty vectors for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (B) HepG2 SR and Huh7 SR cells were transfected with PD-L1 shRNA or empty vectors for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (C) HepG2 SR and Huh7 SR cells were treated with decitabine for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (D) HepG2 SR and Huh7 SR cells were treated with Bay 11 for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (E) Resistant cells were treated with DMSO, decitabine, Bay 11, or decitabine plus Bay 11 for 6 h then subjected to colony-forming assay. Representative images of colony-forming assay (left) and the quantification of colonies (right). Data are mean ± SD, *P

    Journal: Oncology Reports

    Article Title: Targeting the PD-L1/DNMT1 axis in acquired resistance to sorafenib in human hepatocellular carcinoma

    doi: 10.3892/or.2017.5722

    Figure Lengend Snippet: Genetic or pharmacological disruption of PD-L1 or DNMT1 sensitizes HCC resistance to sorafenib. (A) HepG2 SR and Huh7 SR cells were transfected with DNMT1 shRNA or empty vectors for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (B) HepG2 SR and Huh7 SR cells were transfected with PD-L1 shRNA or empty vectors for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (C) HepG2 SR and Huh7 SR cells were treated with decitabine for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (D) HepG2 SR and Huh7 SR cells were treated with Bay 11 for 12 h, treated with sorafenib for additional 72 h and subjected to CCK-8 assays. (E) Resistant cells were treated with DMSO, decitabine, Bay 11, or decitabine plus Bay 11 for 6 h then subjected to colony-forming assay. Representative images of colony-forming assay (left) and the quantification of colonies (right). Data are mean ± SD, *P

    Article Snippet: DNMT3a and DNMT3b have mostly de novo DNA methylation activity, whereas DNMT1 plays a central role in preserving the patterns of DNA methylation through cell division ( ).

    Techniques: Transfection, shRNA, CCK-8 Assay

    PD-L1 induces DNMT1-dependent DNA hypomethylation and restores the expression of methylation-silenced CDH1. (A) GEO data were analyzed for CDH1 expression in sorafenib-sensitive mice versus sorafenib-resistant mice. (B) qPCR measuring the expression levels of CDH1 genes in HepG2 C and Huh7 C vs HepG2 SR and Huh7 SR cells. (C) qPCR for CDH1 expression in HepG2 SR and Huh7 SR cells transfected with empty or PD-L1 shRNA. (D) Bisulfite analysis for the change of DNA methylation in CDH1 promoter (transcription start site −251 to +139) in HepG2 SR cells transfected with empty or PD-L1 shRNA. Data are mean ± SD, *P

    Journal: Oncology Reports

    Article Title: Targeting the PD-L1/DNMT1 axis in acquired resistance to sorafenib in human hepatocellular carcinoma

    doi: 10.3892/or.2017.5722

    Figure Lengend Snippet: PD-L1 induces DNMT1-dependent DNA hypomethylation and restores the expression of methylation-silenced CDH1. (A) GEO data were analyzed for CDH1 expression in sorafenib-sensitive mice versus sorafenib-resistant mice. (B) qPCR measuring the expression levels of CDH1 genes in HepG2 C and Huh7 C vs HepG2 SR and Huh7 SR cells. (C) qPCR for CDH1 expression in HepG2 SR and Huh7 SR cells transfected with empty or PD-L1 shRNA. (D) Bisulfite analysis for the change of DNA methylation in CDH1 promoter (transcription start site −251 to +139) in HepG2 SR cells transfected with empty or PD-L1 shRNA. Data are mean ± SD, *P

    Article Snippet: DNMT3a and DNMT3b have mostly de novo DNA methylation activity, whereas DNMT1 plays a central role in preserving the patterns of DNA methylation through cell division ( ).

    Techniques: Expressing, Methylation, Mouse Assay, Real-time Polymerase Chain Reaction, Transfection, shRNA, DNA Methylation Assay

    PD-L1 regulates DNMT1 through STAT3 signaling. (A) Western blotting (left) and qPCR (right) for DNMT1 and PD-L1 in HepG2 SR transfected with PD-L1 empty vector or shRNA. (B and C) Western blotting for the protein expression of p-STAT3 and total STAT3 in HepG2 SR and HepG2 C (B) or HepG2 SR transfected with PDL1 empty vector or shRNA (C). (D) HepG2 SR cells were transfected with STAT3 siRNA or its control vector for 48 h and subjected to ChIP. The change of STAT3 binding on DNMT1 promoter was assessed by qPCR. (E) 293T cells were transfected with pGL3-DNMT1 alone or plus STAT3 vectors for 48 h, followed by the measurement of luciferase activity. Data are mean ± SD, *P

    Journal: Oncology Reports

    Article Title: Targeting the PD-L1/DNMT1 axis in acquired resistance to sorafenib in human hepatocellular carcinoma

    doi: 10.3892/or.2017.5722

    Figure Lengend Snippet: PD-L1 regulates DNMT1 through STAT3 signaling. (A) Western blotting (left) and qPCR (right) for DNMT1 and PD-L1 in HepG2 SR transfected with PD-L1 empty vector or shRNA. (B and C) Western blotting for the protein expression of p-STAT3 and total STAT3 in HepG2 SR and HepG2 C (B) or HepG2 SR transfected with PDL1 empty vector or shRNA (C). (D) HepG2 SR cells were transfected with STAT3 siRNA or its control vector for 48 h and subjected to ChIP. The change of STAT3 binding on DNMT1 promoter was assessed by qPCR. (E) 293T cells were transfected with pGL3-DNMT1 alone or plus STAT3 vectors for 48 h, followed by the measurement of luciferase activity. Data are mean ± SD, *P

    Article Snippet: DNMT3a and DNMT3b have mostly de novo DNA methylation activity, whereas DNMT1 plays a central role in preserving the patterns of DNA methylation through cell division ( ).

    Techniques: Western Blot, Real-time Polymerase Chain Reaction, Transfection, Plasmid Preparation, shRNA, Expressing, Chromatin Immunoprecipitation, Binding Assay, Luciferase, Activity Assay

    HDAC inhibitors down-regulate DNMT1 expression and DNMT enzymatic activity. NT-2 cells were either not treated (Control) or treated with 5 μM MS-275 (48 h), 5 μM MS-IN (48 h), 0.3 μM TSA (24 h), or 5 mM VPA (24 h). After treatment,

    Journal: Molecular Pharmacology

    Article Title: The Reelin and GAD67 Promoters Are Activated by Epigenetic Drugs That Facilitate the Disruption of Local Repressor Complexes

    doi: 10.1124/mol.108.051763

    Figure Lengend Snippet: HDAC inhibitors down-regulate DNMT1 expression and DNMT enzymatic activity. NT-2 cells were either not treated (Control) or treated with 5 μM MS-275 (48 h), 5 μM MS-IN (48 h), 0.3 μM TSA (24 h), or 5 mM VPA (24 h). After treatment,

    Article Snippet: We used specific siRNAs to target knockdown of DNMT1, DNMT3A, or DNMT3B proteins.

    Techniques: Expressing, Activity Assay, Mass Spectrometry

    siRNA-mediated DNMT knock-down induces overexpression of the nontargeted DNMT proteins. siRNA pools (15 nM) targeting DNMT1, DNMT3A, and DNMT3B were transfected into NT-2 cells either individually or in different combinations. For all experiments,

    Journal: Molecular Pharmacology

    Article Title: The Reelin and GAD67 Promoters Are Activated by Epigenetic Drugs That Facilitate the Disruption of Local Repressor Complexes

    doi: 10.1124/mol.108.051763

    Figure Lengend Snippet: siRNA-mediated DNMT knock-down induces overexpression of the nontargeted DNMT proteins. siRNA pools (15 nM) targeting DNMT1, DNMT3A, and DNMT3B were transfected into NT-2 cells either individually or in different combinations. For all experiments,

    Article Snippet: We used specific siRNAs to target knockdown of DNMT1, DNMT3A, or DNMT3B proteins.

    Techniques: Over Expression, Transfection

    DNMT1 was degraded upon S G treatment and the degradation could be blocked by a proteasomal inhibitor

    Journal: Cancer research

    Article Title: 6-Thioguanine Reactivates Epigenetically Silenced Genes in Acute Lymphoblastic Leukemia Cells by Facilitating Proteasome-mediated Degradation of DNMT1

    doi: 10.1158/0008-5472.CAN-10-3430

    Figure Lengend Snippet: DNMT1 was degraded upon S G treatment and the degradation could be blocked by a proteasomal inhibitor

    Article Snippet: Cell extracts were subjected to Western blot analysis; antibodies that specifically recognized human DNMT1 (New England Biolabs), DNMT1-K142me , LSD1 (Cell Signaling, Danvers, MA), β-actin (Abcam, Cambridge, MA) were used at 1:3000, 1:1000, 1:10,000 and 1:10,000 dilutions, respectively.

    Techniques:

    Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535

    Journal: Biochemistry

    Article Title: Maintenance DNA methyltransferase activity in the presence of oxidized forms of 5-methylcytosine: structural basis for ten eleven translocation-mediated DNA demethylation

    doi: 10.1021/acs.biochem.8b00683

    Figure Lengend Snippet: Molecular dynamics (MD) simulations demonstrate an incremental spatial displacement of oxo-mC from the TRD hydrophobic binding pocket. A . Residues Cys1501, Leu1502, and Met1535 make up the target recognition domain (TRD) and harbor the methyl group of mC, providing the specificity of DNMT1 for hemi-methylated DNA. The MD simulations quantify the displacement of the oxidized forms of mC from these residues in the TRD: B. Cys1501 C. Leu1502 D. Met1535

    Article Snippet: The TRD of human DNMT1 contains a hydrophobic binding pocket consisting of M1535, C1501, L1502 and W1512.

    Techniques: Binding Assay, Methylation