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<p><small><strong> Figure 1 ChIP results obtained with the Diagenode antibody directed against MBD1</strong><br /> ChIP assays were performed using human osteosarcoma (U2OS) cells, the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) and optimized PCR primer sets. Sheared chromatin from 1x10e6 cells and 1.5 μg of antibody were used per ChIP experiment. Beads only were used as a negative IP control. Quantitative PCR was performed with primers for the promoters of the MLH1 gene (used as a positive control) and CDC6 gene (used as a negative control). Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
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<p><small><strong> Figure 2 Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against human MBD1 (Cat. No. pAb-078-050), crude serum and Flow Through. The plates were coated with the peptide used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the purified antibody was estimated to be 1:20,000. </small></p>
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<p><small><strong> Figure 3 Western blot analysis using the Diagenode antibody directed against MBD1</strong><br /> Nuclear extracts of HeLa cells (40 μg) were analysed by Western blot using the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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<p><small><strong> Figure 1 ChIP results obtained with the Diagenode antibody directed against MBD1</strong><br /> ChIP assays were performed using human osteosarcoma (U2OS) cells, the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) and optimized PCR primer sets. Sheared chromatin from 1x10e6 cells and 1.5 μg of antibody were used per ChIP experiment. Beads only were used as a negative IP control. Quantitative PCR was performed with primers for the promoters of the MLH1 gene (used as a positive control) and CDC6 gene (used as a negative control). Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
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<p><small><strong> Figure 2 Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against human MBD1 (Cat. No. pAb-078-050), crude serum and Flow Through. The plates were coated with the peptide used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the purified antibody was estimated to be 1:20,000. </small></p>
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<p><small><strong> Figure 3 Western blot analysis using the Diagenode antibody directed against MBD1</strong><br /> Nuclear extracts of HeLa cells (40 μg) were analysed by Western blot using the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
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<div class="small-2 columns"><img src="https://www.diagenode.com/emailing/images/epi-success-guaranteed-icon.png" alt="Epigenetic success guaranteed" /></div>
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<p><strong>ChIP results</strong> obtained with the antibody directed against H3K4me3 (Cat. No. <a href="../p/h3k4me3-polyclonal-antibody-premium-50-ug-50-ul">C15410003</a>). </p>
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<p>Our aim at Diagenode is to offer the largest collection of highly specific <strong>ChIP-grade antibodies</strong>. We add new antibodies monthly. Find your ChIP-grade antibody in the list below and check more information about tested applications, extensive validation data, and product information.</p>',
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'description' => '<p style="text-align: justify;"><span>Epigenetic research tools have evolved over time from endpoint PCR to qPCR to the analyses of large sets of genome-wide sequencing data. ChIP sequencing (ChIP-seq) has now become the gold standard method for chromatin studies, given the accuracy and coverage scale of the approach over other methods. Successful ChIP-seq, however, requires a higher level of experimental accuracy and consistency in all steps of ChIP than ever before. Particularly crucial is the quality of ChIP antibodies. </span></p>',
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'name' => 'Novel role of UHRF1 in the epigenetic repression of the latent HIV-1.',
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'description' => '<p>BACKGROUND: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC). METHODS: We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4 T-cell models and ex vivo cultures of PBMCs from HIV individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24 protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA. FINDINGS: We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations. INTERPRETATION: A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies. FUNDING: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin », the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation »), « Les Amis des Instituts Pasteur à Bruxelles, asbl », the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the "Alsace contre le Cancer" Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.</p>',
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'description' => '<p>The H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG-binding domain protein 1 MBD1 as a physical and functional partner of the H19 long noncoding RNA (lncRNA). The H19 lncRNA-MBD1 complex is required for the control of five genes of the IGN. For three of these genes-Igf2 (insulin-like growth factor 2), Slc38a4 (solute carrier family 38 member 4), and Peg1 (paternally expressed gene 1)-both MBD1 and H3K9me3 binding were detected on their differentially methylated regions. The H19 lncRNA-MBD1 complex, through its interaction with histone lysine methyltransferases, therefore acts by bringing repressive histone marks on the differentially methylated regions of these three direct targets of the H19 gene. Our data suggest that, besides the differential DNA methylation found on the differentially methylated regions of imprinted genes, an additional fine tuning of the expressed allele is achieved by a modulation of the H3K9me3 marks, mediated by the association of the H19 lncRNA with chromatin-modifying complexes, such as MBD1. This results in a precise control of the level of expression of growth factors in the embryo.</p>',
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'description' => '<p><span>Alternative names: <strong>CXXC3</strong>, <strong>PCM1</strong>, <strong>RFT</strong></span></p>
<p><span>Polyclonal antibody raised in rabbit against human MBD1 (Methyl-CpG-binding domain protein 1), using a KLH-conjugated synthetic peptide containing a sequence from the N-terminal part of the protein.</span></p>',
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15410078_fig3.jpg" alt="MBD1 Antibody ChIP Grade" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 1 ChIP results obtained with the Diagenode antibody directed against MBD1</strong><br /> ChIP assays were performed using human osteosarcoma (U2OS) cells, the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) and optimized PCR primer sets. Sheared chromatin from 1x10e6 cells and 1.5 μg of antibody were used per ChIP experiment. Beads only were used as a negative IP control. Quantitative PCR was performed with primers for the promoters of the MLH1 gene (used as a positive control) and CDC6 gene (used as a negative control). Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
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<p><small><strong> Figure 2 Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against human MBD1 (Cat. No. pAb-078-050), crude serum and Flow Through. The plates were coated with the peptide used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the purified antibody was estimated to be 1:20,000. </small></p>
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<p><small><strong> Figure 3 Western blot analysis using the Diagenode antibody directed against MBD1</strong><br /> Nuclear extracts of HeLa cells (40 μg) were analysed by Western blot using the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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<td>ChIP <sup>*</sup></td>
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<p><span>Polyclonal antibody raised in rabbit against human MBD1 (Methyl-CpG-binding domain protein 1), using a KLH-conjugated synthetic peptide containing a sequence from the N-terminal part of the protein.</span></p>',
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15410078_fig3.jpg" alt="MBD1 Antibody ChIP Grade" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 1 ChIP results obtained with the Diagenode antibody directed against MBD1</strong><br /> ChIP assays were performed using human osteosarcoma (U2OS) cells, the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) and optimized PCR primer sets. Sheared chromatin from 1x10e6 cells and 1.5 μg of antibody were used per ChIP experiment. Beads only were used as a negative IP control. Quantitative PCR was performed with primers for the promoters of the MLH1 gene (used as a positive control) and CDC6 gene (used as a negative control). Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
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<p><img src="https://www.diagenode.com/img/product/antibodies/C15410078_fig1.jpg" alt="MBD1 Antibody ELISA validation" style="display: block; margin-left: auto; margin-right: auto;" /></p>
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<p><small><strong> Figure 2 Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against human MBD1 (Cat. No. pAb-078-050), crude serum and Flow Through. The plates were coated with the peptide used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the purified antibody was estimated to be 1:20,000. </small></p>
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<p><small><strong> Figure 3 Western blot analysis using the Diagenode antibody directed against MBD1</strong><br /> Nuclear extracts of HeLa cells (40 μg) were analysed by Western blot using the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
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<li>Batch-specific data is available on the website</li>
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<p><br />Chromatin immunoprecipitation (<b>ChIP</b>) is a technique to study the associations of proteins with the specific genomic regions in intact cells. One of the most important steps of this protocol is the immunoprecipitation of targeted protein using the antibody specifically recognizing it. The quality of antibodies used in ChIP is essential for the success of the experiment. Diagenode offers extensively validated ChIP-grade antibodies, confirmed for their specificity, and high level of performance in ChIP. Each batch is validated, and batch-specific data are available on the website.</p>
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<div class="small-2 columns"><img src="https://www.diagenode.com/emailing/images/epi-success-guaranteed-icon.png" alt="Epigenetic success guaranteed" /></div>
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<p><strong>ChIP results</strong> obtained with the antibody directed against H3K4me3 (Cat. No. <a href="../p/h3k4me3-polyclonal-antibody-premium-50-ug-50-ul">C15410003</a>). </p>
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<div class="small-12 medium-6 large-6 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15410003-fig1-ChIP.jpg" alt="" width="400" height="315" /> </div>
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<p></p>
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<p>Our aim at Diagenode is to offer the largest collection of highly specific <strong>ChIP-grade antibodies</strong>. We add new antibodies monthly. Find your ChIP-grade antibody in the list below and check more information about tested applications, extensive validation data, and product information.</p>',
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'authors' => 'Verdikt R. et al. ',
'description' => '<p>BACKGROUND: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC). METHODS: We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4 T-cell models and ex vivo cultures of PBMCs from HIV individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24 protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA. FINDINGS: We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations. INTERPRETATION: A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies. FUNDING: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin », the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation »), « Les Amis des Instituts Pasteur à Bruxelles, asbl », the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the "Alsace contre le Cancer" Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.</p>',
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'description' => '<p>The H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG-binding domain protein 1 MBD1 as a physical and functional partner of the H19 long noncoding RNA (lncRNA). The H19 lncRNA-MBD1 complex is required for the control of five genes of the IGN. For three of these genes-Igf2 (insulin-like growth factor 2), Slc38a4 (solute carrier family 38 member 4), and Peg1 (paternally expressed gene 1)-both MBD1 and H3K9me3 binding were detected on their differentially methylated regions. The H19 lncRNA-MBD1 complex, through its interaction with histone lysine methyltransferases, therefore acts by bringing repressive histone marks on the differentially methylated regions of these three direct targets of the H19 gene. Our data suggest that, besides the differential DNA methylation found on the differentially methylated regions of imprinted genes, an additional fine tuning of the expressed allele is achieved by a modulation of the H3K9me3 marks, mediated by the association of the H19 lncRNA with chromatin-modifying complexes, such as MBD1. This results in a precise control of the level of expression of growth factors in the embryo.</p>',
'date' => '2013-12-17',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/24297921',
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'description' => '<p>The H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG-binding domain protein 1 MBD1 as a physical and functional partner of the H19 long noncoding RNA (lncRNA). The H19 lncRNA-MBD1 complex is required for the control of five genes of the IGN. For three of these genes-Igf2 (insulin-like growth factor 2), Slc38a4 (solute carrier family 38 member 4), and Peg1 (paternally expressed gene 1)-both MBD1 and H3K9me3 binding were detected on their differentially methylated regions. The H19 lncRNA-MBD1 complex, through its interaction with histone lysine methyltransferases, therefore acts by bringing repressive histone marks on the differentially methylated regions of these three direct targets of the H19 gene. Our data suggest that, besides the differential DNA methylation found on the differentially methylated regions of imprinted genes, an additional fine tuning of the expressed allele is achieved by a modulation of the H3K9me3 marks, mediated by the association of the H19 lncRNA with chromatin-modifying complexes, such as MBD1. This results in a precise control of the level of expression of growth factors in the embryo.</p>',
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<p><small><strong> Figure 1 ChIP results obtained with the Diagenode antibody directed against MBD1</strong><br /> ChIP assays were performed using human osteosarcoma (U2OS) cells, the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) and optimized PCR primer sets. Sheared chromatin from 1x10e6 cells and 1.5 μg of antibody were used per ChIP experiment. Beads only were used as a negative IP control. Quantitative PCR was performed with primers for the promoters of the MLH1 gene (used as a positive control) and CDC6 gene (used as a negative control). Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
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<p><small><strong> Figure 2 Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against human MBD1 (Cat. No. pAb-078-050), crude serum and Flow Through. The plates were coated with the peptide used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the purified antibody was estimated to be 1:20,000. </small></p>
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<p><small><strong> Figure 3 Western blot analysis using the Diagenode antibody directed against MBD1</strong><br /> Nuclear extracts of HeLa cells (40 μg) were analysed by Western blot using the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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<td>Western Blotting</td>
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<td>Fig 3</td>
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<p><span>Polyclonal antibody raised in rabbit against human MBD1 (Methyl-CpG-binding domain protein 1), using a KLH-conjugated synthetic peptide containing a sequence from the N-terminal part of the protein.</span></p>',
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<p><small><strong> Figure 1 ChIP results obtained with the Diagenode antibody directed against MBD1</strong><br /> ChIP assays were performed using human osteosarcoma (U2OS) cells, the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) and optimized PCR primer sets. Sheared chromatin from 1x10e6 cells and 1.5 μg of antibody were used per ChIP experiment. Beads only were used as a negative IP control. Quantitative PCR was performed with primers for the promoters of the MLH1 gene (used as a positive control) and CDC6 gene (used as a negative control). Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
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<p><small><strong> Figure 2 Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against human MBD1 (Cat. No. pAb-078-050), crude serum and Flow Through. The plates were coated with the peptide used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the purified antibody was estimated to be 1:20,000. </small></p>
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<p><small><strong> Figure 3 Western blot analysis using the Diagenode antibody directed against MBD1</strong><br /> Nuclear extracts of HeLa cells (40 μg) were analysed by Western blot using the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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<td>ChIP <sup>*</sup></td>
<td>1.5 μg/ChIP</td>
<td>Fig 1</td>
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<tr>
<td>ELISA</td>
<td>1:1,000</td>
<td>Fig 2</td>
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<tr>
<td>Western Blotting</td>
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<td>Fig 3</td>
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<p><small><sup>*</sup> Please note that the optimal antibody amount per IP should be determined by the end-user. We recommend testing 1-5 μg per IP.</small></p>',
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<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
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<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
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<li>Batch-specific data is available on the website</li>
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'meta_title' => 'Diagenode's selection of Antibodies is exclusively dedicated for Epigenetic Research | Diagenode',
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<p><br />Chromatin immunoprecipitation (<b>ChIP</b>) is a technique to study the associations of proteins with the specific genomic regions in intact cells. One of the most important steps of this protocol is the immunoprecipitation of targeted protein using the antibody specifically recognizing it. The quality of antibodies used in ChIP is essential for the success of the experiment. Diagenode offers extensively validated ChIP-grade antibodies, confirmed for their specificity, and high level of performance in ChIP. Each batch is validated, and batch-specific data are available on the website.</p>
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<div class="small-2 columns"><img src="https://www.diagenode.com/emailing/images/epi-success-guaranteed-icon.png" alt="Epigenetic success guaranteed" /></div>
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<p><strong>ChIP results</strong> obtained with the antibody directed against H3K4me3 (Cat. No. <a href="../p/h3k4me3-polyclonal-antibody-premium-50-ug-50-ul">C15410003</a>). </p>
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<div class="small-12 medium-6 large-6 columns"><img src="https://www.diagenode.com/img/product/antibodies/C15410003-fig1-ChIP.jpg" alt="" width="400" height="315" /> </div>
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<p>Our aim at Diagenode is to offer the largest collection of highly specific <strong>ChIP-grade antibodies</strong>. We add new antibodies monthly. Find your ChIP-grade antibody in the list below and check more information about tested applications, extensive validation data, and product information.</p>',
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'description' => '<p>BACKGROUND: The multiplicity, heterogeneity, and dynamic nature of human immunodeficiency virus type-1 (HIV-1) latency mechanisms are reflected in the current lack of functional cure for HIV-1. Accordingly, all classes of latency-reversing agents (LRAs) have been reported to present variable ex vivo potencies. Here, we investigated the molecular mechanisms underlying the potency variability of one LRA: the DNA methylation inhibitor 5-aza-2'-deoxycytidine (5-AzadC). METHODS: We employed epigenetic interrogation methods (electrophoretic mobility shift assays, chromatin immunoprecipitation, Infinium array) in complementary HIV-1 infection models (latently-infected T-cell line models, primary CD4 T-cell models and ex vivo cultures of PBMCs from HIV individuals). Extracellular staining of cell surface receptors and intracellular metabolic activity were measured in drug-treated cells. HIV-1 expression in reactivation studies was explored by combining the measures of capsid p24 protein, green fluorescence protein signal, intracellular and extracellular viral RNA and viral DNA. FINDINGS: We uncovered specific demethylation CpG signatures induced by 5-AzadC in the HIV-1 promoter. By analyzing the binding modalities to these CpG, we revealed the recruitment of the epigenetic integrator Ubiquitin-like with PHD and RING finger domain 1 (UHRF1) to the HIV-1 promoter. We showed that UHRF1 redundantly binds to the HIV-1 promoter with different binding modalities where DNA methylation was either non-essential, essential or enhancing UHRF1 binding. We further demonstrated the role of UHRF1 in the epigenetic repression of the latent viral promoter by a concerted control of DNA and histone methylations. INTERPRETATION: A better understanding of the molecular mechanisms of HIV-1 latency allows for the development of innovative antiviral strategies. As a proof-of-concept, we showed that pharmacological inhibition of UHRF1 in ex vivo HIV patient cell cultures resulted in potent viral reactivation from latency. Together, we identify UHRF1 as a novel actor in HIV-1 epigenetic silencing and highlight that it constitutes a new molecular target for HIV-1 cure strategies. FUNDING: Funding was provided by the Belgian National Fund for Scientific Research (F.R.S.-FNRS, Belgium), the « Fondation Roi Baudouin », the NEAT (European AIDS Treatment Network) program, the Internationale Brachet Stiftung, ViiV Healthcare, the Télévie, the Walloon Region (« Fonds de Maturation »), « Les Amis des Instituts Pasteur à Bruxelles, asbl », the University of Brussels (Action de Recherche Concertée ULB grant), the Marie Skodowska Curie COFUND action, the European Union's Horizon 2020 research and innovation program under grant agreement No 691119-EU4HIVCURE-H2020-MSCA-RISE-2015, the French Agency for Research on AIDS and Viral Hepatitis (ANRS), the Sidaction and the "Alsace contre le Cancer" Foundation. This work is supported by 1UM1AI164562-01, co-funded by National Heart, Lung and Blood Institute, National Institute of Diabetes and Digestive and Kidney Diseases, National Institute of Neurological Disorders and Stroke, National Institute on Drug Abuse and the National Institute of Allergy and Infectious Diseases.</p>',
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'description' => '<p>The H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG-binding domain protein 1 MBD1 as a physical and functional partner of the H19 long noncoding RNA (lncRNA). The H19 lncRNA-MBD1 complex is required for the control of five genes of the IGN. For three of these genes-Igf2 (insulin-like growth factor 2), Slc38a4 (solute carrier family 38 member 4), and Peg1 (paternally expressed gene 1)-both MBD1 and H3K9me3 binding were detected on their differentially methylated regions. The H19 lncRNA-MBD1 complex, through its interaction with histone lysine methyltransferases, therefore acts by bringing repressive histone marks on the differentially methylated regions of these three direct targets of the H19 gene. Our data suggest that, besides the differential DNA methylation found on the differentially methylated regions of imprinted genes, an additional fine tuning of the expressed allele is achieved by a modulation of the H3K9me3 marks, mediated by the association of the H19 lncRNA with chromatin-modifying complexes, such as MBD1. This results in a precise control of the level of expression of growth factors in the embryo.</p>',
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'name' => 'H19 lncRNA controls gene expression of the Imprinted Gene Network by recruiting MBD1.',
'authors' => 'Monnier P, Martinet C, Pontis J, Stancheva I, Ait-Si-Ali S, Dandolo L',
'description' => '<p>The H19 gene controls the expression of several genes within the Imprinted Gene Network (IGN), involved in growth control of the embryo. However, the underlying mechanisms of this control remain elusive. Here, we identified the methyl-CpG-binding domain protein 1 MBD1 as a physical and functional partner of the H19 long noncoding RNA (lncRNA). The H19 lncRNA-MBD1 complex is required for the control of five genes of the IGN. For three of these genes-Igf2 (insulin-like growth factor 2), Slc38a4 (solute carrier family 38 member 4), and Peg1 (paternally expressed gene 1)-both MBD1 and H3K9me3 binding were detected on their differentially methylated regions. The H19 lncRNA-MBD1 complex, through its interaction with histone lysine methyltransferases, therefore acts by bringing repressive histone marks on the differentially methylated regions of these three direct targets of the H19 gene. Our data suggest that, besides the differential DNA methylation found on the differentially methylated regions of imprinted genes, an additional fine tuning of the expressed allele is achieved by a modulation of the H3K9me3 marks, mediated by the association of the H19 lncRNA with chromatin-modifying complexes, such as MBD1. This results in a precise control of the level of expression of growth factors in the embryo.</p>',
'date' => '2013-12-17',
'pmid' => 'https://www.ncbi.nlm.nih.gov/pubmed/24297921',
'doi' => '10.1073/pnas.1310201110',
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<p><small><strong> Figure 2 Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against human MBD1 (Cat. No. pAb-078-050), crude serum and Flow Through. The plates were coated with the peptide used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the purified antibody was estimated to be 1:20,000. </small></p>
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<p><small><strong> Figure 3 Western blot analysis using the Diagenode antibody directed against MBD1</strong><br /> Nuclear extracts of HeLa cells (40 μg) were analysed by Western blot using the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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<p><small><strong> Figure 1 ChIP results obtained with the Diagenode antibody directed against MBD1</strong><br /> ChIP assays were performed using human osteosarcoma (U2OS) cells, the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) and optimized PCR primer sets. Sheared chromatin from 1x10e6 cells and 1.5 μg of antibody were used per ChIP experiment. Beads only were used as a negative IP control. Quantitative PCR was performed with primers for the promoters of the MLH1 gene (used as a positive control) and CDC6 gene (used as a negative control). Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). </small></p>
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<p><small><strong> Figure 2 Determination of the antibody titer</strong><br /> To determine the titer of the antibody, an ELISA was performed using a serial dilution of the Diagenode antibody directed against human MBD1 (Cat. No. pAb-078-050), crude serum and Flow Through. The plates were coated with the peptide used for immunization of the rabbit. By plotting the absorbance against the antibody dilution (Figure 2), the titer of the purified antibody was estimated to be 1:20,000. </small></p>
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<p><small><strong> Figure 3 Western blot analysis using the Diagenode antibody directed against MBD1</strong><br /> Nuclear extracts of HeLa cells (40 μg) were analysed by Western blot using the Diagenode antibody against MBD1 (Cat. No. pAb-078-050) diluted 1:500 in TBS-Tween containing 5% skimmed milk. The position of the protein of interest is indicated on the right, the marker (in kDa) is shown on the left. </small></p>
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<p>Learn more about: <a href="https://www.diagenode.com/applications/western-blot">Loading control, MW marker visualization</a><em>. <br /></em></p>
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'description' => '<p><span style="font-weight: 400;">All Diagenode’s antibodies are listed below. Please, use our Quick search field to find the antibody of interest by target name, application, purity.</span></p>
<p><span style="font-weight: 400;">Diagenode’s highly validated antibodies:</span></p>
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<li>Highly sensitive and specific</li>
<li>Cost-effective (requires less antibody per reaction)</li>
<li>Batch-specific data is available on the website</li>
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<div class="small-10 columns"><center></center>
<p><br />Chromatin immunoprecipitation (<b>ChIP</b>) is a technique to study the associations of proteins with the specific genomic regions in intact cells. One of the most important steps of this protocol is the immunoprecipitation of targeted protein using the antibody specifically recognizing it. The quality of antibodies used in ChIP is essential for the success of the experiment. Diagenode offers extensively validated ChIP-grade antibodies, confirmed for their specificity, and high level of performance in ChIP. Each batch is validated, and batch-specific data are available on the website.</p>
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<div class="small-2 columns"><img src="https://www.diagenode.com/emailing/images/epi-success-guaranteed-icon.png" alt="Epigenetic success guaranteed" /></div>
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<p><strong>ChIP results</strong> obtained with the antibody directed against H3K4me3 (Cat. No. <a href="../p/h3k4me3-polyclonal-antibody-premium-50-ug-50-ul">C15410003</a>). </p>
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<p>Our aim at Diagenode is to offer the largest collection of highly specific <strong>ChIP-grade antibodies</strong>. We add new antibodies monthly. Find your ChIP-grade antibody in the list below and check more information about tested applications, extensive validation data, and product information.</p>',
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