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Immunoprecipitation Analysis (immunoprecipitation + analysis)

Distribution by Scientific Domains
Life Sciences55%
Medical Sciences44%

Kinds of Immunoprecipitation Analysis

  • chromatin immunoprecipitation analysis
    		•

    Selected Abstracts

    IL-1, induces stabilization of IL-8 mRNA in malignant breast cancer cells via the 3, untranslated region: Involvement of divergent RNA-binding factors HuR, KSRP and TIAR,

    INTERNATIONAL JOURNAL OF CANCER, Issue 6 2005
    Esther A. Suswam
    Abstract IL-8 plays an integral role in promoting the malignant phenotype in breast cancer, and its production is directly influenced by inflammatory cytokines in the tumor microenvironment. Here, we show that activation of IL-1, receptors on malignant HS578t and MDA-MB-231 breast cancer cells strongly induces IL-8 expression and that RNA stabilization is persistently activated at least 12,24 hr after stimulation. SB 203580 and rapamycin reversed the RNA stabilization effect of IL-1, in a dose-dependent manner, suggesting involvement of the p38/MAP kinase and mTOR pathways. A luciferase reporter assay indicated that the stabilization effect was dependent on cis elements in the 3,-untranslated region (UTR) of the IL-8 transcript. By UV cross-linking, we identified multiple cellular factors that interact with the IL-8 3,UTR, ranging 34,76 kDa. Immunoprecipitation analysis indicated that HuR, KSRP and TIAR bound to one or more loci in the 3,UTR. While the cross-linking patterns were similar, quantitative immunoprecipitation of native IL-8 RNA from IL-1,-stimulated cytoplasmic extract revealed a 20-fold greater association of transcript with the stabilizing factor HuR vs. the destabilizing factor KSRP. In conclusion, IL-1, is a potent cytokine stimulus for IL-8 RNA stabilization in breast cancer cells, possibly by enhanced binding of cytoplasmic HuR to the 3,UTR. Published 2004 Wiley-Liss, Inc. [source]

    Two short-chain dehydrogenase/reductases, NON-YELLOW COLORING 1 and NYC1-LIKE, are required for chlorophyll b and light-harvesting complex II degradation during senescence in rice

    THE PLANT JOURNAL, Issue 1 2009
    Yutaka Sato
    Summary Yellowing, which is related to the degradation of chlorophyll and chlorophyll,protein complexes, is a notable phenomenon during leaf senescence. NON-YELLOW COLORING1 (NYC1) in rice encodes a membrane-localized short-chain dehydrogenase/reductase (SDR) that is thought to represent a chlorophyll b reductase necessary for catalyzing the first step of chlorophyll b degradation. Analysis of the nyc1 mutant, which shows the stay-green phenotype, revealed that chlorophyll b degradation is required for the degradation of light-harvesting complex II and thylakoid grana in leaf senescence. Phylogenetic analysis further revealed the existence of NYC1-LIKE (NOL) as the most closely related protein to NYC1. In the present paper, the nol mutant in rice was also found to show a stay-green phenotype very similar to that of the nyc1 mutant, i.e. the degradation of chlorophyll b was severely inhibited and light-harvesting complex II was selectively retained during senescence, resulting in the retention of thylakoid grana even at a late stage of senescence. The nyc1 nol double mutant did not show prominent enhancement of inhibition of chlorophyll degradation. NOL was localized on the stromal side of the thylakoid membrane despite the lack of a transmembrane domain. Immunoprecipitation analysis revealed that NOL and NYC1 interact physically in vitro. These observations suggest that NOL and NYC1 are co-localized in the thylakoid membrane and act in the form of a complex as a chlorophyll b reductase in rice. [source]

    Jarid2 is among a set of genes differentially regulated by Nkx2.5 during outflow tract morphogenesis

    DEVELOPMENTAL DYNAMICS, Issue 7 2010
    Jeremy L. Barth
    Abstract Nkx2.5, a transcription factor implicated in human congenital heart disease, is required for regulation of second heart field (SHF) progenitors contributing to outflow tract (OFT). Here, we define a set of genes (Lrrn1, Elovl2, Safb, Slc39a6, Khdrbs1, Hoxb4, Fez1, Ccdc117, Jarid2, Nrcam, and Enpp3) expressed in SHF containing pharyngeal arch tissue whose regulation is dependent on Nkx2.5. Further investigation shows that Jarid2, which has been implicated in OFT morphogenesis, is a direct target of Nkx2.5 regulation. Jarid2 expression was up-regulated in SHF mesoderm of Nkx2.5-deficient embryos. Chromatin immunoprecipitation analysis showed Nkx2.5 interaction with consensus binding sites in the Jarid2 promoter in pharyngeal arch cells. Finally, Jarid2 promoter activity and mRNA expression levels were down-regulated by Nkx2.5 overexpression. Given the role of Jarid2 as a regulator of early cardiac proliferation, these findings highlight Jarid2 as one of several potential mediators of the critical role played by Nkx2.5 during OFT morphogenesis. Developmental Dynamics 239:2024,2033, 2010. © 2010 Wiley-Liss, Inc. [source]

    DPPA4 modulates chromatin structure via association with DNA and core histone H3 in mouse embryonic stem cells

    GENES TO CELLS, Issue 4 2010
    Hisaharu Masaki
    Developmental pluripotency associated 4 (DPPA4) is one of the uncharacterized genes that is highly expressed in embryonic stem (ES) cells. DPPA4 is associated with active chromatin and involved in the pluripotency of mouse ES cells. However, the biological function of DPPA4 remains poorly understood. In this study, we performed fluorescence recovery after photobleaching (FRAP) analysis to examine the dynamics of DPPA4 in ES cells. FRAP analysis showed that the mobility of DPPA4 is similar to that of histone H1. In addition, biochemical analysis with purified proteins and immunoprecipitation analysis showed that DPPA4 directly binds to both DNA and core histone H3. The analysis using truncated proteins indicated that DPPA4 is associated with DNA via the N-terminal region and histone H3 via the C-terminal region. In vitro assembled chromatin showed resistance to micrococcal nuclease (MNase) digestion in the presence of DPPA4. Moreover, MNase assay and FRAP analysis with the truncated proteins implies that DPPA4 binding to both DNA and histone H3 is necessary for the chromatin structure resistant to MNase and for the proper localization of DPPA4 in ES cell nuclei. These results suggest that DPPA4 modulates the chromatin structure in association with DNA and histone H3 in ES cells. [source]

    Transcription regulation of the Saccharomyces cerevisiae PIS1 gene by inositol and the pleiotropic regulator, Ume6p

    MOLECULAR MICROBIOLOGY, Issue 6 2008
    Niketa M. Jani
    Summary In Saccharomyces cerevisiae, transcription of most of the phospholipid biosynthetic genes (e.g. INO1, CHO1, CHO2 and OPI3) is repressed by growth in the presence of inositol and choline and derepressed in their absence. This regulation requires the Ino2p and Ino4p activators and the Opi1p repressor. The PIS1 structural gene is required for the synthesis of the essential lipid phosphatidylinositol. Previous reports show that PIS1 expression is uncoupled from inositol/choline regulation, but is regulated by carbon source, hypoxia and zinc. However, in this study we found that the expression of PIS1 is induced twofold by inositol. This regulation did not require Ino2p and Ino4p, although Ino4p was required for full expression. Ino4p is a basic helix-loop-helix protein that requires a binding partner. Curiously, none of the other basic helix-loop-helix proteins affected PIS1 expression. Inositol induction did require another general regulator of phospholipid biosynthesis, Ume6p. Ume6p was found to be a positive regulator of PIS1 gene expression. Ume6p, and several associated factors, were required for inositol-mediated induction and chromatin immunoprecipitation analysis showed that Ume6p directly regulates PIS1 expression. Thus, we demonstrate novel regulation of the PIS1 gene by Ume6p. [source]

    Down-Regulation of Melanin Synthesis by a Biphenyl Derivative and Its Mechanism

    PIGMENT CELL & MELANOMA RESEARCH, Issue 5 2003
    Kyoko Nakamura
    Down-regulation of melanin synthesis is required for recovery of pigmentary disorders and it is known that direct inhibitors of tyrosinase, the key enzyme in melanin synthesis, such as hydroquinone with a phenol structure, suppress melanin synthesis. We screened several phenolic derivatives using B16 melanoma cells and found that a biphenyl derivative, 2,2,-dihydroxy-5,5,-dipropyl-biphenyl (DDB), down-regulated melanin synthesis effectively. Although DDB has a phenol structure, it did not inhibit tyrosinase in vitro, thus we examined its mechanism in detail. Western blotting revealed that the amount of tyrosinase was decreased by DDB, and pulse-chase labeling and immunoprecipitation analysis showed a decrease of mature tyrosinase and acceleration of tyrosinase degradation in its presence. These results suggest that DDB down-regulates melanin synthesis by inhibiting the maturation of tyrosinase, leading to acceleration of tyrosinase degradation. [source]

    Smad3 signalling plays an important role in keloid pathogenesis via epithelial,mesenchymal interactions

    THE JOURNAL OF PATHOLOGY, Issue 2 2005
    TT Phan
    Abstract Smad signalling plays important roles in developmental and cancer biology as well as in fibropathogenesis. Its role in keloid biology is not known. Epithelial,mesenchymal interactions, originally described in normal skin, have recently been established to play a significant role in keloid pathogenesis, and demonstrate the important influence of keratinocyte paracrine factor signalling on fibroblast behaviour. The present study investigated the role of downstream Smad cascade induction in this interaction. Normal fibroblasts (NF) and keloid fibroblasts (KF) were co-cultured in serum-free medium with normal keratinocytes (NK) or keloid keratinocytes (KK) for 5 days, after which fibroblast cell lysates were subjected to western blot and immunoprecipitation analysis to quantify the levels of Smad and Smad2/3/4 binding complex. In another set of experiments, wild-type (wt), Smad2-null (Smad2,/,) and Smad3-null (Smad3,/,) mouse embryonic fibroblasts (MEF) were assayed for cell proliferation and collagen production after serum-free co-culture with KK or exposure to conditioned media collected from serum-free KK/KF co-culture. Compared to normal skin, keloids expressed high basal levels of TGF,R1 and TGF,R2, Smad2, 3 and 4 and phospho-Smad2. Upregulation of TGF,R1 and TGF,R2, Smad3 and p-Smad2 was observed in KF co-cultured with KK, together with enhanced Smad3 phosphorylation and Smad2/3/4 binding complex production. When MEF-wt, MEF-Smad2,/, or MEF-Smad3,/, were co-cultured with KK or exposed to KK/KF co-culture conditioned media, enhanced proliferation and collagen production were seen in MEF-wt and MEF-Smad2,/, but not in MEF-Smad3,/, cells. The activation of Smad signalling, importantly that of Smad3, appears to be one facet of the complex epithelial,mesenchymal interactions in keloid pathogenesis, resulting in active KF proliferation and collagen-ECM production in co-culture with KK. This finding suggests the suppression of Smad signalling as a novel approach in keloid therapy. Copyright © 2005 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [source]

    Differential mechanism of NF-,B inhibition by two glucocorticoid receptor modulators in rheumatoid arthritis synovial fibroblasts

    ARTHRITIS & RHEUMATISM, Issue 11 2009
    Valerie Gossye
    Objective To investigate and compare the molecular mechanisms by which 2 glucocorticoid receptor (GR),activating compounds, dexamethasone (DEX) and Compound A (CpdA), interfere with the NF-,B activation pathway in rheumatoid arthritis (RA) synovial cells. Methods Quantitative polymerase chain reaction was performed to detect the tumor necrosis factor , (TNF,),induced cytokine gene expression of interleukin-1, (IL-1,) and to investigate the effects of DEX and CpdA in RA fibroblast-like synoviocytes (FLS) transfected with small interfering RNA (siRNA) against GR (siGR) compared with nontransfected cells. Immunofluorescence analysis was used to detect the subcellular distribution of NF-,B (p65) under the various treatment conditions, and active DNA-bound p65 was measured using a TransAM assay and by chromatin immunoprecipitation analysis of IL-1,. Signaling pathways were studied via Western blotting of siGR-transfected cells, compared with nontransfected and nontargeting siRNA,transfected control cells, to detect the regulation of phospho-IKK, I,B,, phospho-p38, phospho-ERK, and phospho-JNK. Results Both DEX and CpdA efficiently inhibited IL-1, gene expression in a GR-dependent manner. In addition, CpdA attenuated the TNF,-induced nuclear translocation and DNA binding of p65 in RA FLS, via the attenuation of IKK phosphorylation and subsequent I,B, degradation. CpdA also displayed profound effects on TNF,-induced MAPK activation. The effects of CpdA on TNF,-induced kinase activities occurred independently of the presence of GR. In sharp contrast, DEX did not affect TNF,-induced IKK phosphorylation, I,B, degradation, p65 nuclear translocation, or MAPK activation in RA FLS. Conclusion DEX and CpdA display a dissimilar molecular mechanism of interaction with the NF-,B activation pathway ex vivo. A dual pathway, partially dependent and partially independent of GR (nongenomic), may explain the gene-inhibitory effects of CpdA in RA FLS. [source]

    Human ovarian carcinoma cells: Histone deacetylase inhibitors exhibit antiproliferative activity and potently induce apoptosis

    CANCER, Issue 12 2004
    Noriyuki Takai M.D., Ph.D.
    Abstract BACKGROUND Histone deacetylase inhibitors (HDACIs) can inhibit proliferation, stimulate apoptosis, and induce cell cycle arrest in malignant cells. METHODS The authors investigated the effects of four HDACIs on nine ovarian carcinoma cell lines in vitro and in vivo. Ovarian carcinoma cells were treated with a variety of HDACIs, and their effects on cell growth, the cell cycle, apoptosis, and related events were investigated. The ability of valproic acid (VPA) to inhibit the growth of ovarian tumors in immunodeficient mice was also assessed. RESULTS Clonogenic assays showed that all ovarian carcinoma cell lines were sensitive to the growth-inhibitory effects of the HDACIs. Cell cycle analysis indicated that their exposure to HDACIs decreased the proportion of cells in S phase and increased the proportion of cells in the G0/G1 and/or G2/M phases of the cell cycle. Terminal deoxynucleotidyltransferase-mediated uridine triphosphate end-labeling assays demonstrated that HDACIs induced apoptosis, which occurred in concert with alterations in the expression of genes related to apoptosis, cell growth, and malignant phenotype, including the activation of caspase-9 and caspase-3. Chromatin immunoprecipitation analysis revealed a notable increase in levels of acetylated histones associated with the p21 promoter after treatment with suberoylanilide bishydroxamine. In addition, in experiments involving nude mice, VPA significantly inhibited human ovarian tumor growth without toxic side effects. CONCLUSIONS The results of the current study suggest that HDACIs may be particularly effective in the treatment of ovarian tumors. Cancer 2004. © 2004 American Cancer Society. [source]