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  • 92
    Santa Cruz Biotechnology anti mbd1
    Localization of endogenous <t>Mbd1</t> in DNA methylation-deficient cells. (A) Immunocytochemistry of Dnmt1 +/+ (left panels) or Dnmt1 n/n (right panels) cells as determined using anti-Mbd1 antibodies (top panels). DNA was counter stained using DAPI (bottom panels). (B) Southern blot of DNA from Dnmt1 +/+ and Dnmt1 n/n cells. TaiI-digested genomic DNA was blotted and probed with a major satellite (sat) sequence. TaiI cleaves at the sequence ACGT and is blocked by methylation of CpG.
    Anti Mbd1, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    86
    Intel pentium m 24 5 w processor
    Localization of endogenous <t>Mbd1</t> in DNA methylation-deficient cells. (A) Immunocytochemistry of Dnmt1 +/+ (left panels) or Dnmt1 n/n (right panels) cells as determined using anti-Mbd1 antibodies (top panels). DNA was counter stained using DAPI (bottom panels). (B) Southern blot of DNA from Dnmt1 +/+ and Dnmt1 n/n cells. TaiI-digested genomic DNA was blotted and probed with a major satellite (sat) sequence. TaiI cleaves at the sequence ACGT and is blocked by methylation of CpG.
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    Image Search Results


    Localization of endogenous Mbd1 in DNA methylation-deficient cells. (A) Immunocytochemistry of Dnmt1 +/+ (left panels) or Dnmt1 n/n (right panels) cells as determined using anti-Mbd1 antibodies (top panels). DNA was counter stained using DAPI (bottom panels). (B) Southern blot of DNA from Dnmt1 +/+ and Dnmt1 n/n cells. TaiI-digested genomic DNA was blotted and probed with a major satellite (sat) sequence. TaiI cleaves at the sequence ACGT and is blocked by methylation of CpG.

    Journal: Molecular and Cellular Biology

    Article Title: Mbd1 Is Recruited to both Methylated and Nonmethylated CpGs via Distinct DNA Binding Domains

    doi: 10.1128/MCB.24.8.3387-3395.2004

    Figure Lengend Snippet: Localization of endogenous Mbd1 in DNA methylation-deficient cells. (A) Immunocytochemistry of Dnmt1 +/+ (left panels) or Dnmt1 n/n (right panels) cells as determined using anti-Mbd1 antibodies (top panels). DNA was counter stained using DAPI (bottom panels). (B) Southern blot of DNA from Dnmt1 +/+ and Dnmt1 n/n cells. TaiI-digested genomic DNA was blotted and probed with a major satellite (sat) sequence. TaiI cleaves at the sequence ACGT and is blocked by methylation of CpG.

    Article Snippet: Slides were blocked in 3% bovine serum albumin-PBS before incubation with primary antibody (anti-Flag M2 [Stratagene], anti-Gal4 [Santa Cruz], or anti-Mbd1 [M245] [Santa Cruz] [all at 1:1,000]) for 60 min.

    Techniques: DNA Methylation Assay, Immunocytochemistry, Staining, Southern Blot, Sequencing, Methylation

    The CXXC-3 domain of Mbd1 recognizes nonmethylated CpG. (A) Alignment of the three CXXC domains (CXXC-1, CXXC-2, and CXXC-3) from Mbd1 with the CXXC domains from DNMT1, MLL, and CGBP. The consensus sequence of the domain is given above. An asterisk marks cysteine C356 that is mutated in the CXXC-3-C356A peptide. (B and C) Band shifts with probes containing nonmethylated (CG), methylated (mCG), or hemimethylated CG or a probe lacking CG entirely (TG). (B) Increasing amounts (50, 250, or 750 ng) of recombinant wild-type CXXC-3 (CxxC-3) or mutant CXXC-3 (CxxC-3-C356A; 750 ng) peptide were added. (C) Increasing amounts (100, 500, or 1,000 ng) of a peptide spanning CXXC-1 and CXXC-2 (CxxC-1/2) or CXXC-3 (250 ng) were added. (D) Increasing amounts (50, 200, or 500 ng) of recombinant wild-type (MBD) or mutant (MBD-R22A; 500 ng) MBD peptide were added.

    Journal: Molecular and Cellular Biology

    Article Title: Mbd1 Is Recruited to both Methylated and Nonmethylated CpGs via Distinct DNA Binding Domains

    doi: 10.1128/MCB.24.8.3387-3395.2004

    Figure Lengend Snippet: The CXXC-3 domain of Mbd1 recognizes nonmethylated CpG. (A) Alignment of the three CXXC domains (CXXC-1, CXXC-2, and CXXC-3) from Mbd1 with the CXXC domains from DNMT1, MLL, and CGBP. The consensus sequence of the domain is given above. An asterisk marks cysteine C356 that is mutated in the CXXC-3-C356A peptide. (B and C) Band shifts with probes containing nonmethylated (CG), methylated (mCG), or hemimethylated CG or a probe lacking CG entirely (TG). (B) Increasing amounts (50, 250, or 750 ng) of recombinant wild-type CXXC-3 (CxxC-3) or mutant CXXC-3 (CxxC-3-C356A; 750 ng) peptide were added. (C) Increasing amounts (100, 500, or 1,000 ng) of a peptide spanning CXXC-1 and CXXC-2 (CxxC-1/2) or CXXC-3 (250 ng) were added. (D) Increasing amounts (50, 200, or 500 ng) of recombinant wild-type (MBD) or mutant (MBD-R22A; 500 ng) MBD peptide were added.

    Article Snippet: Slides were blocked in 3% bovine serum albumin-PBS before incubation with primary antibody (anti-Flag M2 [Stratagene], anti-Gal4 [Santa Cruz], or anti-Mbd1 [M245] [Santa Cruz] [all at 1:1,000]) for 60 min.

    Techniques: Sequencing, Methylation, Recombinant, Mutagenesis

    Localization of Mbd1 in DNA methylation-deficient cells requires the CXXC-3 domain. (A) Schematic representation of the Flag-Mbd1a expression constructs employed. The positions of the MBD (cross-hatched), CXXC-3 (dark gray), and the TRD (hatched) are shown. The CXXC-1 and CXXC-2 positions are shown shaded in light gray, and the Flag tag position is dotted. An asterisk marks the point mutations in the MBD (R22A), and “Δ” marks the sites of deletion of CXXC-3 or the TRD. (B) Diagram illustrating the enrichment for methyl CpG or nonmethyl CpG in the heterochromatic regions of wild-type ( Dnmt1 +/+ ) or methylation-deficient ( Dnmt1 n/n ) cells, respectively. Methyl-CpG is shown with filled circles and nonmethyl-CpGs with open circles. The heterochromatic regions are represented by lightly shaded boxes. (C) Dnmt1 +/+ (panels a to d) or Dnmt1 n/n (panels e to h) mouse embryonic fibroblasts were transfected with constructs expressing Flag-tagged Mbd1a (either full length or the indicated mutants). At 48 h after transfection, the cells were fixed and stained using an anti-Flag antibody (left panels). DNA was counterstained with DAPI (right panels). (D) Schematic of Gal4-DBD-MBD1 fusion constructs analyzed for colocalization with heterochromatic foci in Dnmt1 +/+ or Dnmt1 n/n mouse embryonic fibroblasts. A plus sign indicates colocalization with heterochromatic foci in Dnmt1 n/n cells and diffuse staining in Dnmt1 +/+ cells, whereas a minus sign indicates diffuse staining in cells of either genotype. The fusion constructs encode amino acids 81 to 556, 261 to 556, and 261 to 346 of the MBD1 isoform PCM1. The Gal4-DBD is shown in black. CXXC-2 is shown shaded in light gray, CXXC-3 is shown shaded in dark gray, and the TRD is hatched. (Lower panels) Colocalization with heterochromatic foci of the Gal4-DBD-MBD1 (amino acids 261 to 346) (Gal4-DBD-CXXC-3) in Dnmt1 n/n cells is shown.

    Journal: Molecular and Cellular Biology

    Article Title: Mbd1 Is Recruited to both Methylated and Nonmethylated CpGs via Distinct DNA Binding Domains

    doi: 10.1128/MCB.24.8.3387-3395.2004

    Figure Lengend Snippet: Localization of Mbd1 in DNA methylation-deficient cells requires the CXXC-3 domain. (A) Schematic representation of the Flag-Mbd1a expression constructs employed. The positions of the MBD (cross-hatched), CXXC-3 (dark gray), and the TRD (hatched) are shown. The CXXC-1 and CXXC-2 positions are shown shaded in light gray, and the Flag tag position is dotted. An asterisk marks the point mutations in the MBD (R22A), and “Δ” marks the sites of deletion of CXXC-3 or the TRD. (B) Diagram illustrating the enrichment for methyl CpG or nonmethyl CpG in the heterochromatic regions of wild-type ( Dnmt1 +/+ ) or methylation-deficient ( Dnmt1 n/n ) cells, respectively. Methyl-CpG is shown with filled circles and nonmethyl-CpGs with open circles. The heterochromatic regions are represented by lightly shaded boxes. (C) Dnmt1 +/+ (panels a to d) or Dnmt1 n/n (panels e to h) mouse embryonic fibroblasts were transfected with constructs expressing Flag-tagged Mbd1a (either full length or the indicated mutants). At 48 h after transfection, the cells were fixed and stained using an anti-Flag antibody (left panels). DNA was counterstained with DAPI (right panels). (D) Schematic of Gal4-DBD-MBD1 fusion constructs analyzed for colocalization with heterochromatic foci in Dnmt1 +/+ or Dnmt1 n/n mouse embryonic fibroblasts. A plus sign indicates colocalization with heterochromatic foci in Dnmt1 n/n cells and diffuse staining in Dnmt1 +/+ cells, whereas a minus sign indicates diffuse staining in cells of either genotype. The fusion constructs encode amino acids 81 to 556, 261 to 556, and 261 to 346 of the MBD1 isoform PCM1. The Gal4-DBD is shown in black. CXXC-2 is shown shaded in light gray, CXXC-3 is shown shaded in dark gray, and the TRD is hatched. (Lower panels) Colocalization with heterochromatic foci of the Gal4-DBD-MBD1 (amino acids 261 to 346) (Gal4-DBD-CXXC-3) in Dnmt1 n/n cells is shown.

    Article Snippet: Slides were blocked in 3% bovine serum albumin-PBS before incubation with primary antibody (anti-Flag M2 [Stratagene], anti-Gal4 [Santa Cruz], or anti-Mbd1 [M245] [Santa Cruz] [all at 1:1,000]) for 60 min.

    Techniques: DNA Methylation Assay, Expressing, Construct, FLAG-tag, Methylation, Transfection, Staining

    Mbd1 isoforms in mouse. (A) Schematic of the murine Mbd1 locus showing the differentially spliced exons (exons 10, 15, and 15a) shaded in gray. (B) Alignment of the C termini of the predicted MBD1 proteins from the human MBD1v1 (hMBD1), rat MBD1 (rMBD1), and murine Mbd1a/b (mMBD1a) and Mbd1c/d (mMBD1c) cDNA. (C) Schematic representation of the predicted Mbd1 proteins in mouse. CXXC domains 1 and 2 are shown shaded in light grey; the differentially spliced CXXC-3 domain is shaded in dark gray. The MBD (cross-hatched) and TRD (hatched) are indicated, and the novel alternative C terminus is shaded in black. (D) Mbd1a and Mbd1c isoforms both repress a methylated reporter gene. HeLa cells were transfected with the methylated pGL2 reporter and expression vectors for Mbd1a or Mbd1c. Means and standard deviations of the luciferase activity of triplicate transfections from a representative experiment are shown. The value for the activity without an Mbd1 expression vector is arbitrarily set to 1. (E) A Western blot (WB) of in vitro transcription and translation reactions of the possible splice variants alongside a sample of nuclear extract (NE) from murine fibroblasts. Bands in the NE lane are somewhat bowed due to the high protein concentration and are therefore aligned at their leading edges. The asterisk marks a cross-reacting band that is not Mbd1.

    Journal: Molecular and Cellular Biology

    Article Title: Mbd1 Is Recruited to both Methylated and Nonmethylated CpGs via Distinct DNA Binding Domains

    doi: 10.1128/MCB.24.8.3387-3395.2004

    Figure Lengend Snippet: Mbd1 isoforms in mouse. (A) Schematic of the murine Mbd1 locus showing the differentially spliced exons (exons 10, 15, and 15a) shaded in gray. (B) Alignment of the C termini of the predicted MBD1 proteins from the human MBD1v1 (hMBD1), rat MBD1 (rMBD1), and murine Mbd1a/b (mMBD1a) and Mbd1c/d (mMBD1c) cDNA. (C) Schematic representation of the predicted Mbd1 proteins in mouse. CXXC domains 1 and 2 are shown shaded in light grey; the differentially spliced CXXC-3 domain is shaded in dark gray. The MBD (cross-hatched) and TRD (hatched) are indicated, and the novel alternative C terminus is shaded in black. (D) Mbd1a and Mbd1c isoforms both repress a methylated reporter gene. HeLa cells were transfected with the methylated pGL2 reporter and expression vectors for Mbd1a or Mbd1c. Means and standard deviations of the luciferase activity of triplicate transfections from a representative experiment are shown. The value for the activity without an Mbd1 expression vector is arbitrarily set to 1. (E) A Western blot (WB) of in vitro transcription and translation reactions of the possible splice variants alongside a sample of nuclear extract (NE) from murine fibroblasts. Bands in the NE lane are somewhat bowed due to the high protein concentration and are therefore aligned at their leading edges. The asterisk marks a cross-reacting band that is not Mbd1.

    Article Snippet: Slides were blocked in 3% bovine serum albumin-PBS before incubation with primary antibody (anti-Flag M2 [Stratagene], anti-Gal4 [Santa Cruz], or anti-Mbd1 [M245] [Santa Cruz] [all at 1:1,000]) for 60 min.

    Techniques: Methylation, Transfection, Expressing, Luciferase, Activity Assay, Plasmid Preparation, Western Blot, In Vitro, Protein Concentration