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  • Over-accumulation of nuclear IGF-1 receptor in tumor cells requires elevated expression of the receptor and the SUMO-conjugating enzyme Ubc9. 21147068

    The insulin-like growth factor 1 receptor (IGF-1R) plays crucial roles in tumor cell growth and is overexpressed in many cancers. IGF-1R's trans-membrane kinase signaling pathways have been well characterized. Very recently, we showed that SUMOylation mediates nuclear translocation of the IGF-1R, and that nuclear IGF-1R (nIGF-1R) binds to enhancer regions and activates transcription. We identified three lysine residues in the ?-subunit of the receptor and that mutation of these blocks nuclear translocation and gene activation. Furthermore, accumulation of nIGF-1R was proven strongly dependent on the specific SUMO-conjugating enzyme Ubc9. Here we show that nIGF-1R originates solely from the cell membrane and that phosphorylation of the core tyrosine residues of the receptor kinase is crucial for nuclear accumulation. We also compared the levels of nIGF-1R, measured as nuclear/membrane ratios, in tumor and normal cells. We found that the breast cancer cell line MCF-7 has 13-fold higher amounts of nIGF-1R than breast epithelial cells (IME) which showed only a small amount of nIGF-1R. In comparison, the total expression of IGF-1R was only 3.7- higher in MCF-7. Comparison of several other tumor and normal cell lines showed similar tumor cell over-accumulation of nIGF-1R, exceeding the total receptor expression substantially. Ectopic overexpression (>10-fold) of the receptor increased nIGF-1R in IME cells but not to that high level as in wild type MCF-7. The levels of Ubc9 were higher in all tumor cell lines, compared to the normal cells, and this probably contributes to over-accumulation of nIGF-1R. Over-accumulation of nIGF-1R may contribute to deregulated gene expression and therewith play a pathophysiological role in cancer cells.
    Tipo de documento:
    Referencia
    Referencia del producto:
    LP1
    Nombre del producto:
    VLDL, human

  • A novel mechanism for target gene-specific SWI/SNF recruitment via the Snf2p N-terminus. 21278159

    Chromatin-remodeling complexes regulate the expression of genes in all eukaryotic genomes. The SWI/SNF complex of Saccharomyces cerevisiae is recruited to its target promoters via interactions with selected transcription factors. Here, we show that the N-terminus of Snf2p, the chromatin remodeling core unit of the SWI/SNF complex, is essential for the expression of VHT1, the gene of the plasma membrane H(+)/biotin symporter, and of BIO5, the gene of a 7-keto-8-aminopelargonic acid transporter, biotin biosynthetic precursor. chromatin immunoprecipitation (ChIP) analyses demonstrate that Vhr1p, the transcriptional regulator of VHT1 and BIO5 expression, is responsible for the targeting of Snf2p to the VHT1 promoter at low biotin. We identified an Snf2p mutant, Snf2p-R(15)C, that specifically abolishes the induction of VHT1 and BIO5 but not of other Snf2p-regulated genes, such as GAL1, SUC2 or INO1. We present a novel mechanism of target gene-specific SWI/SNF recruitment via Vhr1p and a conserved N-terminal Snf2p domain.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB3580
    Nombre del producto:
    Anti-Green Fluorescent Protein Antibody

  • Characterization and functional analysis of the 5'-flanking promoter region of the mouse Tcf3 gene. 21935611

    Tcf3 is a nuclear mediator of canonical Wnt signaling which functions in many systems as a repressor of target gene transcription. In this study, we have cloned and characterized a 6.7 kb fragment of the 5'-flanking promoter region of the mouse Tcf3 gene. In silico analysis of the promoter sequence identified the existence of GC boxes and CpG islands, but failed to identify any TATA box. In addition, the promoter sequence contained putative binding sites for multiple transcription factors, including a few known to regulate the function of mTcf3. Of those, we confirmed functional binding sites for NF?B and Oct1 using a luciferase assay and ChIP. In vitro analysis revealed five potential transcription start sites which resulted in a 298 base pair 5'-untranslated region upstream of the mTcf3 translation start site ATG. Using a luciferase assay, we analyzed the activity of the cloned promoter fragment in embryonically derived neural stem cells. The luciferase activity of a 3.5 kb core promoter fragment (-3243/+211) showed up to 40-fold increased activity compared to the empty vector. Addition of sequences 5' to the 3.5 kb core promoter fragment resulted in a 20-fold drop in luciferase activity, indicating the presence of further upstream repressive elements. In vivo analysis of a 4.5 kb promoter fragment (-4303/+211) driving, the expression of EGFP in mouse embryos highly resembled endogenous expression of mTcf3.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB5434

  • The histone demethylase JARID1A regulates progesterone receptor expression. 21348942

    Transcriptional control of the progesterone receptor gene by estrogen is a complex mechanism. It involves estrogen receptor α which uses several enzymes that locally modify histone tails as cofactors. Using MCF-7 cells as a model, we found that Jumonji AT-rich interactive domain 1A (JARID1A; KDM5A/RBP2), an enzyme that removes the activating H3K4 di- and trimethylation marks, was involved in the fine-tuning of progesterone receptor gene expression. Reduction of JARID1A led to enhanced progesterone receptor expression, at both the basal and estrogen-stimulated levels. Conversely, overexpression of JARID1A wild-type, but not the enzymatically inactive mutant, suppressed progesterone receptor promoter activity. Chromatin immunoprecipitation experiments showed JARID1A to bind in a ligand-independent manner to a progesterone receptor gene upstream region that contains an estrogen response element half-site as well as the CCGCCC sequence, which is potentially recognized by JARID1A. Estrogen treatment led to RNA polymerase II recruitment to this region and to increased estrogen receptor α binding to the PR enhancer region 1. In addition, elevation of H3K4 trimethylation was detected at the estrogen response element half-site region. Reduction of JARID1A expression was followed by higher H3K4 trimethylation in this region. Analysis of MDA-MB-231 cells, which do not express the progesterone receptor, indicated that H3K4 trimethylation did not take place in the regulatory regions examined. Taken together, the results underscore the importance of epigenetic modifications for regulation of progesterone receptor expression. They suggest that H3K4 tri- and dimethylation play an important role and that JARID1A is the histone-demethylating enzyme responsible for removal of this mark.
    Tipo de documento:
    Referencia
    Referencia del producto:
    PP64
    Nombre del producto:
    IgG, Rabbit

  • BioVyon Protein A, an alternative solid-phase affinity matrix for chromatin immunoprecipitation. 21284925

    Chromatin immunoprecipitation (ChIP) is an important technique in the study of DNA/protein interactions. The ChIP procedure, however, has limitations in that it is lengthy, can be inconsistent, and is prone to nonspecific binding of DNA and proteins to the bead-based solid-phase matrices that are often used for the immunoprecipitation step. In this investigation, we examined the utility of a new matrix for ChIP assays, BioVyon Protein A, a solid support based on porous polyethylene. In ChIP experiments carried out using two antibodies and seven DNA loci, the performance of BioVyon Protein A was significantly better, with a greater percentage of DNA pull-down in all of the assays tested compared with bead-based matrices, Protein A Sepharose, and Dynabeads Protein A. Furthermore, the rigid porous disc format within a column made the BioVyon matrix much easier to use with fewer steps and less equipment requirements, resulting in a significant reduction in the time taken to process the ChIP samples. In summary, BioVyon Protein A provides a column-based assay method for ChIP and other immunoprecipitation-based procedures; the rigid porous structure of BioVyon enables a fast and robust protocol with higher ChIP enrichment ratios.Crown Copyright © 2011. Published by Elsevier Inc. All rights reserved.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-030
    Nombre del producto:
    Anti-dimethyl-Histone H3 (Lys4) Antibody

  • XRCC1 suppresses somatic hypermutation and promotes alternative nonhomologous end joining in Igh genes. 21967769

    Activation-induced deaminase (AID) deaminates cytosine to uracil in immunoglobulin genes. Uracils in DNA can be recognized by uracil DNA glycosylase and abasic endonuclease to produce single-strand breaks. The breaks are repaired either faithfully by DNA base excision repair (BER) or mutagenically to produce somatic hypermutation (SHM) and class switch recombination (CSR). To unravel the interplay between repair and mutagenesis, we decreased the level of x-ray cross-complementing 1 (XRCC1), a scaffold protein involved in BER. Mice heterozygous for XRCC1 showed a significant increase in the frequencies of SHM in Igh variable regions in Peyer's patch cells, and of double-strand breaks in the switch regions during CSR. Although the frequency of CSR was normal in Xrcc1(+/-) splenic B cells, the length of microhomology at the switch junctions decreased, suggesting that XRCC1 also participates in alternative nonhomologous end joining. Furthermore, Xrcc1(+/-) B cells had reduced Igh/c-myc translocations during CSR, supporting a role for XRCC1 in microhomology-mediated joining. Our results imply that AID-induced single-strand breaks in Igh variable and switch regions become substrates simultaneously for BER and mutagenesis pathways.
    Tipo de documento:
    Referencia
    Referencia del producto:
    12-371
    Nombre del producto:
    Normal Mouse IgG

  • Chromatin state signatures associated with tissue-specific gene expression and enhancer activity in the embryonic limb. 22421546

    The regulatory elements that direct tissue-specific gene expression in the developing mammalian embryo remain largely unknown. Although chromatin profiling has proven to be a powerful method for mapping regulatory sequences in cultured cells, chromatin states characteristic of active developmental enhancers have not been directly identified in embryonic tissues. Here we use whole-transcriptome analysis coupled with genome-wide profiling of H3K27ac and H3K27me3 to map chromatin states and enhancers in mouse embryonic forelimb and hindlimb. We show that gene-expression differences between forelimb and hindlimb, and between limb and other embryonic cell types, are correlated with tissue-specific H3K27ac signatures at promoters and distal sites. Using H3K27ac profiles, we identified 28,377 putative enhancers, many of which are likely to be limb specific based on strong enrichment near genes highly expressed in the limb and comparisons with tissue-specific EP300 sites and known enhancers. We describe a chromatin state signature associated with active developmental enhancers, defined by high levels of H3K27ac marking, nucleosome displacement, hypersensitivity to sonication, and strong depletion of H3K27me3. We also find that some developmental enhancers exhibit components of this signature, including hypersensitivity, H3K27ac enrichment, and H3K27me3 depletion, at lower levels in tissues in which they are not active. Our results establish histone modification profiling as a tool for developmental enhancer discovery, and suggest that enhancers maintain an open chromatin state in multiple embryonic tissues independent of their activity level.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-449
    Nombre del producto:
    Anti-trimethyl-Histone H3 (Lys27) Antibody

  • Mutational analysis reveals a noncontractile but interactive role of actin and profilin in viral RNA-dependent RNA synthesis. 19710142

    As obligatory parasites, viruses co-opt a variety of cellular functions for robust replication. The expression of the nonsegmented negative-strand RNA genome of respiratory syncytial virus (RSV), a significant pediatric pathogen, absolutely requires actin and is stimulated by the actin-regulatory protein profilin. As actin is a major contractile protein, it was important to determine whether the known functional domains of actin and profilin were important for their ability to activate RSV transcription. Analyses of recombinant mutants in a reconstituted RSV transcription system suggested that the divalent-cation-binding domain of actin is critically needed for binding to the RSV genome template and for the activation of viral RNA synthesis. In contrast, the nucleotide-binding domain and the N-terminal acidic domain were needed neither for template binding nor for transcription. Specific surface residues of actin, required for actin-actin contact during filamentation, were also nonessential for viral transcription. Unlike actin, profilin did not directly bind to the viral template but was recruited by actin. Mutation of the interactive residues of actin or profilin, resulting in the loss of actin-profilin binding, also abolished profilin's ability to stimulate viral transcription. Together, these results suggest that actin acts as a classical transcription factor for the virus by divalent-cation-dependent binding to the viral template and that profilin acts as a transcriptional cofactor, in part by associating with actin. This essential viral role of actin is independent of its contractile cellular role.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB858-3B

  • Loss of striatal dopaminergic terminals during the early stage in response to MPTP injection in C57BL/6 mice. 21185886

    The molecular mechanisms underlying MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-induced dopaminergic (DAergic) neuronal death in vivo are still not fully understood. To investigate the selective DAergic neurotoxicity, we have developed an immunological technique to isolate DAergic synaptosomes from mouse striatal tissues using an antibody against 20 amino acid residues in the extracellular second loop of dopamine transporter (DAT). The DAT protein level in the isolated DAergic synaptosomes was markedly decreased at 16 h after a single injection of 30 mg/kg MPTP, but not in striatal homogenate and crude synaptosomes fraction. GBR-12909, a dopamine uptake inhibitor, completely reversed the MPTP-induced decrease of DAT protein in the DAergic synaptosomes. These results suggest that the isolated DAergic synaptosomes can be useful to identify mechanisms of loss of the nerve terminals.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB369
    Nombre del producto:
    Anti-Dopamine Transporter Antibody, NT, clone DAT-Nt

  • Herpes simplex virus type-2 specific glycoprotein G-2 immunomagnetically captured from HEp-2 infected tissue culture extracts. 15158593

    Monoclonal antibody H1206 anti-HSV-2 gG-2 bound to tosylactivated paramagnetic Dynabeads (Dynal) has been used to isolate HSV-2 type-specific gG-2 from solubilized HEp-2 HSV-2 infected cell extracts. The immunomagnetically captured type-specific glycoprotein reacted strongly with monoclonal antibody H1206 and demonstrated a single band with apparent molecular weight of 100000 (100 kDa) and a doublet band with an apparent molecular weight of 60000-64000 (60-64 kDa). We observed the same exact banding pattern when monoclonal H1206 was immunoblotted with Helix pomatia lectin purified HSV-2 gG-2. The immunomagnetically purified gG-2 was unreactive to monoclonal antibody H1379 anti-HSV-1 gG-1 and four human HSV antibody negative sera. In addition, 20 human HSV antibody positive sera obtained from the Centers for Disease Control (CDC), Atlanta, GA, were used for the evaluation of our methodology. Immunoblotting of the human HSV antibody positive samples were in agreement with the CDC HSV serological designation. Sera characterized by reactivity to the immunomagnetically purified gG-2 in conjunction with Western blot has the potential to be used as a confirmatory serological test or to determine the accuracy of clinical serological immunoassays used to determine HSV-2 seropositivity.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo