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  • Detection of citrulline residues in deiminated proteins on polyvinylidene difluoride membrane. 1524220

    We have developed a new detection method of deiminated proteins on polyvinylidene difluoride membranes. Citrulline residues in enzymatically deiminated histones were modified by incubating with diacetyl monoxime and antipyrine in a strong acid mixture. The products were injected to rabbits, and the antibodies obtained were affinity-purified using a modified citrulline column. Sample proteins blotted to the membrane were modified in a similar manner and incubated successively with the purified antibody and an alkaline phosphatase-conjugated second antibody. Detection was performed using a chemiluminescent substrate. The method enabled detection of 3-10 fmol of citrulline residues dot blotted as deiminated model proteins. It visualized numerous rat pituitary soluble proteins that had been enzymatically deiminated and Western blotted to the membrane. The data suggest usefulness of the method for detecting deiminated proteins regardless of the backbone protein molecules. Search for deiminated proteins on the Western blots of various rat tissue homogenates detected a single band on that of spinal cord, another band on that of uterus, and multiple bands on those of skin and hair root. The bands in the former two tissue homogenates comigrated with glial fibrillary acidic protein and vimentin, respectively.
    Document Type:
    Reference
    Product Catalog Number:
    17-347

  • Down syndrome fibroblasts and mouse Prep1-overexpressing cells display increased sensitivity to genotoxic stress. 20110257

    PREP1 (PKNOX1) maps in the Down syndrome (DS) critical region of chromosome 21, is overexpressed in some DS tissues and might be involved in the DS phenotype. By using fibroblasts from DS patients and by overexpressing Prep1 in F9 teratocarcinoma and Prep1(i/i) MEF to single out the role of the protein, we report that excess Prep1 increases the sensitivity of cells to genotoxic stress and the extent of the apoptosis directly correlates with the level of Prep1. The apoptotic response of Prep1-overexpressing cells is mediated by the pro-apoptotic p53 protein that we show is a direct target of Prep1, as its depletion reverts the apoptotic phenotype. The induction of p53 overcomes the anti-apoptotic role of Bcl-X(L), previously shown to be also a Prep1 target, the levels of which are increased in Prep1-overexpressing cells as well. Our results provide a rationale for the involvement of PREP1 in the apoptotic phenotype of DS tissues and indicate that differences in Prep1 level can have drastic effects.
    Document Type:
    Reference
    Product Catalog Number:
    05-766

  • Bovine Foamy Virus Transactivator (BTas) Interacts with Cellular RelB to Enhance Viral Transcription. 20844054

    Viruses are obligate intracellular parasites that depend on cellular machinery for their efficient transcription and replication. In a previous study we reported that bovine foamy virus (BFV) is able to activate the nuclear factor (NF)-κB pathway through the action of its transactivator (BTas) to enhance viral transcription. However, the mechanism used by NF-κB to enhance BFV transcription remains elusive. To address this question we employed a yeast two-hybrid assay to screen for BTas-interacting proteins. We found that RelB, a member of NF-κB protein family, interacts with BTas. We confirmed the putative RelB-BTas interaction in vitro and in vivo, and identified the protein regions responsible for the RelB-BTas interaction. Using a luciferase reporter assay, we next showed that RelB enhances BFV transcription (BTas-induced LTR transactivation), and that this process requires both localization of the RelB-BTas interaction in the nucleus and the Rel homology domain of RelB. Knockdown of cellular endogenous RelB protein using siRNA significantly attenuated BTas-induced LTR transcription. The results of ChIP analysis showed that endogenous RelB binds to the viral LTR in BFV-infected cells. Together, these results suggest that BFV engages RelB protein as a cotransactivator of BTas to enhance viral transcription. In addition, our findings indicate that BFV infection up-regulates cellular RelB expression through BTas-induced NF-κB activation. Thus, this study demonstrates the existence of a positive feed-back circuit in which BFV utilizes the host's NF-κB pathway through RelB protein for efficient viral transcription.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • Endothelium-specific SIRT1 overexpression inhibits hyperglycemia-induced upregulation of vascular cell senescence. 22744176

    The rapidly increasing prevalence of diabetes mellitus worldwide is one of the most serious and challenging health problems in the 21st century. Mammalian sirtuin 1 (SIRT1) has been shown to decrease high-glucose-induced endothelial cell senescence in vitro and prevent hyperglycemia-induced vascular dysfunction. However, a role for SIRT1 in prevention of hyperglycemia-induced vascular cell senescence in vivo remains unclear. We used endothelium-specific SIRT1 transgenic (SIRT1-Tg) mice and wild-type (WT) mice to construct a 40-week streptozotocin (STZ)-induced diabetic mouse model. In this mode, 42.9% of wild-type (WT) mice and 38.5% of SIRT1-Tg mice were successfully established as diabetic. Forty weeks of hyperglycemia induced significant vascular cell senescence in aortas of mice, as indicated by upregulation of expression of senescence-associated markers including p53, p21 and plasminogen activator inhibitor-1 (PAI-1). However, SIRT1-Tg diabetic mice displayed dramatically decreased expression of p53, p21 and PAI-1 compared with diabetic WT mice. Moreover, manganese superoxide dismutase expression (MnSOD) was significantly downregulated in the aortas of diabetic WT mice, but was preserved in diabetic SIRT1-Tg mice. Furthermore, expression of the oxidative stress adaptor p66Shc was significantly decreased in aortas of SIRT1-Tg diabetic mice compared with WT diabetic mice. Overall, these findings suggest that SIRT1-mediated inhibition of hyperglycemia-induced vascular cell senescence is mediated at least partly through the reduction of oxidative stress.
    Document Type:
    Reference
    Product Catalog Number:
    07-131
    Product Catalog Name:
    Anti-Sirt1(Sir2) Antibody

  • The vitamin D receptor agonist elocalcitol inhibits IL-8-dependent benign prostatic hyperplasia stromal cell proliferation and inflammatory response by targeting the RhoA ... 19107880

    BACKGROUND: Benign prostatic hyperplasia (BPH) is characterized by an important inflammatory component. Stimulation of human prostate stromal cells from BPH tissues with proinflammatory cytokines leads to secretion of IL-8, a chemokine involved in BPH pathogenesis. The vitamin D receptor (VDR) agonist elocalcitol can arrest prostate growth in BPH patients, but its mechanism of action in this pathology is still incompletely understood. METHODS: IL-8 levels were measured by real-time RT-PCR and ELISA. NF-kappaB translocation and COX-2 expression were evaluated by confocal microscopy. RhoA and Rho-kinase (ROCK) gene expression and functional activity were studied by real-time RT-PCR, immuno-kinase assays, Western blot analysis, confocal microscopy, and cell invasion. RESULTS: Stimulation of BPH cells with IL-8 activates the calcium-sensitizing RhoA/ROCK pathway, as demonstrated by the increased membrane translocation of RhoA and by phosphorylation of the ROCK substrate myosin phosphatase target subunit 1 (MYPT-1). In agreement with these data, C3 exoenzyme, a selective RhoA inhibitor, inhibits IL-8-induced invasion of BPH cells. The VDR agonist elocalcitol significantly inhibits IL-8 production by BPH cells stimulated with inflammatory cytokines, and IL-8-induced proliferation of BPH cells. In addition, elocalcitol inhibits IL-8-induced membrane translocation of RhoA and MYPT-1 phosphorylation in BPH cells, and inhibits dose-dependently their IL-8-dependent invasion. The inhibition induced by elocalcitol of IL-8 production by BPH cells is accompanied by decreased COX-2 expression and PGE(2) production and by arrest of NF-kappaB p65 nuclear translocation, associated with inhibition of the RhoA/ROCK pathway. CONCLUSIONS: These data provide a mechanistic explanation for the anti-proliferative and anti-inflammatory properties of elocalcitol in BPH cells. Prostate 69:480-493, 2009. (c) 2008 Wiley-Liss, Inc.
    Document Type:
    Reference
    Product Catalog Number:
    05-773
    Product Catalog Name:
    Anti-phospho-MYPT1 (Thr850) Antibody, clone SA19, rabbit monoclonal

  • Interleukin-2 inhibits glucocorticoid receptor transcriptional activity through a mechanism involving STAT5 (signal transducer and activator of transcription 5) but not A ... 11435608

    Cytokines and glucocorticoids (GCs) signaling pathways interfere with each other in the regulation of apoptosis and gene expression in the immune system. Interleukin-2 (IL-2), through the Janus kinase/signal transducers and activators of transcription (Jak/STAT) and mitogen-activated protein kinase (MAPK) pathways, activates STAT5 and activated protein-1 (AP-1) transcription factors, respectively, which are known to repress glucocorticoid receptor (GR) activity, at least in part, through protein-protein interactions. In this work, we have analyzed the mechanisms whereby IL-2 down-regulates the GC-induced transactivation of the mouse mammary tumor virus long terminal repeat (MMTV-LTR) in murine CTLL-2 T lymphocytes. Mutagenesis studies revealed that the MMTV-LTR STAT5 binding site (-923/-914) was not required for IL-2-mediated inhibition but identified both glucocorticoid response elements (GREs) and the -104/+1 region as critical elements for this negative response. The DNA binding activities of transcription factors required for GC-mediated activation of the MMTV-LTR promoter and that bind to the -104/+1 region (nuclear factor-1, Oct-1) were not affected by IL-2 treatment. Overexpression of wild-type STAT5B enhanced the effect of IL-2 on MMTV-LTR activity, and a dominant negative form of STAT5B (Y699F) abolished the IL-2-mediated MMTV-LTR inhibition, whereas AP-1 activation had no effect in this system. Direct interaction between liganded GR and STAT5 was observed in CTLL-2 cells in a STAT5 phosphorylation-independent manner. Overexpression of nuclear coactivators CBP (CREB-binding protein) or SRC-1a (steroid receptor coactivator 1a) did not blunt IL-2 inhibitory effects. We suggest that the STAT5-repressive activity on the GC-dependent transcription may involve direct interaction of STAT5 with GR, is dependent on the promoter context and STAT5 activation level, and occurs independently of coactivators levels in T cells.
    Document Type:
    Reference
    Product Catalog Number:
    06-969

  • Epigenetic regulation of the stem cell mitogen Fgf-2 by Mbd1 in adult neural stem/progenitor cells. 18689796

    Whether and how mechanisms intrinsic to stem cells modulate their proliferation and differentiation are two central questions in stem cell biology. Although exogenous basic fibroblast growth factor 2 (FGF-2/Fgf-2) is commonly used to expand adult neural stem/progenitor cells (NSPCs) in vitro, we do not yet understand the functional significance or the molecular regulation of Fgf-2 expressed endogenously by adult NSPCs. We previously demonstrated that methylated CpG binding protein 1 (MBD1/Mbd1) is a transcriptional repressor of Fgf-2 and is enriched in adult brains. Mbd1 deficiency in mice selectively affected adult neurogenesis and the differentiation of NSPCs. Here we show that an Mbd1 and DNA methylation-mediated epigenetic mechanism regulated the expression of stem cell mitogen Fgf-2 in adult NSPCs. Mbd1 bound to the Fgf-2 promoter and regulates its expression in adult NSPCs. In the absence of functional Mbd1, the Fgf-2 promoter was hypomethylated, and treatment with a DNA methylation inhibitor resulted in increased Fgf-2 expression in adult NSPCs. We further demonstrated that both acute knockdown of Mbd1 or overexpression of Fgf-2 in adult NSPCs inhibited their neuronal differentiation, which could be responsible for the neurogenic deficits observed in Mbd1-deficient mice. These data indicate that intrinsic epigenetic mechanisms play critical roles in the regulation of adult NSPC functions.
    Document Type:
    Reference
    Product Catalog Number:
    17-295
    Product Catalog Name:
    Chromatin Immunoprecipitation (ChIP) Assay Kit

  • Sarcolemmal Na+/H+ exchanger activity and expression in human ventricular myocardium. 10933369

    OBJECTIVES: To determine sarcolemmal Na+/H+ exchanger (NHE) activity and expression in human ventricular myocardium. BACKGROUND: Although the sarcolemmal NHE has been implicated in various physiological and pathophysiological phenomena in animal studies, its activity and expression in human myocardium have not been studied. METHODS: Ventricular myocardium was obtained from unused donor hearts with acute myocardial dysfunction (n = 5) and recipient hearts with chronic end stage heart failure (n = 11) through a transplantation program. Intracellular pH (pHi) was monitored in enzymatically isolated single ventricular myocytes by microepifluorescence. As the index of sarcolemmal NHE activity, the rate of H+ efflux at a pHi of 6.90 J(H6.9)) was determined after the induction of intracellular acidosis in bicarbonate-free medium. Na+/H+ exchanger isoform 1 (NHE1) expression in ventricular myocardium was determined by immunoblot analysis. RESULTS: Human ventricular myocytes exhibited readily detectable sarcolemmal NHE activity after the induction of intracellular acidosis, and this activity was suppressed by the NHE1-selective inhibitor HOE-642 (cariporide) at 1 micromol/L. Sarcolemmal NHE activity of myocytes was significantly greater in recipient hearts (JH6.9 = 1.95+/-0.18 mmol/L/min) than it was in unused donor hearts (J(H6.9 = 1.06+/-0.15 mmol/L/min). In contrast, NHE1 protein was expressed in similar abundance in ventricular myocardium from both recipient and unused donor hearts. CONCLUSIONS: Sarcolemmal NHE activity of human ventricular myocytes arises from the NHE1 isoform and is inhibited by HOE-642. Sarcolemmal NHE activity is significantly greater in recipient hearts with chronic end-stage heart failure than it is in unused donor hearts, and this difference is likely to arise from altered posttranslational regulation.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3140
    Product Catalog Name:
    Anti-Na+/H+ Exchanger-1 Antibody, CT, clone 4E9

  • Connective tissue growth factor inhibits metastasis and acts as an independent prognostic marker in colorectal cancer. 15633118

    BACKGROUND AIMS: Connective tissue growth factor (CTGF) has been shown to be implicated in tumor development and progression. The aim of this study was to investigate the role of CTGF in progression of colorectal cancer (CRC). METHODS: Immunohistochemical staining of specimens from 119 patients with CRC was performed. Liposome-mediated transfection was used to introduce a CTGF expression vector into CRC cell lines. Transfectants were tested in invasive ability and experimental hepatic metastasis in BALB/c mice. Furthermore, a FOPflash/TOPflash reporter assay was performed to investigate CTGF on the beta-catenin/T-cell factor signaling pathway. RESULTS: Patients with stage II and stage III CRC whose tumors displayed high CTGF expression had a significantly higher overall survival and a disease-free advantage over patients with CRC with low CTGF expression. Alterations in the CTGF level in CRC cell lines modulated their invasive ability with an inverse correlation. In addition, a reduction in the CTGF level of CT26 cells after stable transfection with antisense CTGF resulted in increased liver metastasis in BALB/c mice. The activity of the beta-catenin/T-cell factor signaling pathway and its downstream effector gene matrix metalloproteinase 7 in these CTGF-transfected cells was strongly attenuated. Blockage of matrix metalloproteinase 7 with its neutralizing antibodies inhibited increased invasiveness in antisense CTGF-transfected CT26 cells. CONCLUSIONS: Our results implicate CTGF as a key regulator of CRC invasion and metastasis, and it appears to be a useful and better prognosis factor for patients with stage II and stage III CRC.
    Document Type:
    Reference
    Product Catalog Number:
    MAB3322

  • Nucleolar localization of hepatic c-Myc: a potential mechanism for c-Myc regulation. 15777849

    The c-myc proto-oncogene encodes a transcription factor that is involved in cell proliferation, growth, differentiation, and apoptosis. Previous studies on the regulation of hepatic c-myc have focused on control of its mRNA expression, which generally correlates with hepatocyte proliferation during both liver development and liver regeneration. However, Western blot analysis showed similar levels of hepatic c-Myc in fetal and adult liver. We therefore went on to examine the abundance and distribution of hepatic c-Myc. Immunofluorescence on adult rat liver cryosections showed that c-Myc was readily detectable, but that it was largely localized to the nucleolus. In contrast, proliferating fetal hepatocytes and adult hepatocytes from regenerating liver showed a diffuse nuclear pattern. Transient transfection of adult hepatocytes with full-length HA-Myc also revealed localization to the nucleolus. Western immunoblotting studies confirmed that immunoreactive c-Myc was present in nucleolar extracts isolated from adult liver. We speculate that the nucleolus may act to sequester c-Myc in quiescent hepatocytes while providing a pool of c-Myc that is readily available to reach its targets in the nucleus.
    Document Type:
    Reference
    Product Catalog Number:
    06-340
    Product Catalog Name:
    Anti-Myc Antibody