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  • Neuroprotective role of PrPC against kainate-induced epileptic seizures and cell death depends on the modulation of JNK3 activation by GluR67-PSD-95 binding. 21757544

    Cellular prion protein (PrP(C)) is a glycosyl-phosphatidylinositol-anchored glycoprotein. When mutated or misfolded, the pathogenic form (PrP(SC)) induces transmissible spongiform encephalopathies. In contrast, PrP(C) has a number of physiological functions in several neural processes. Several lines of evidence implicate PrP(C) in synaptic transmission and neuroprotection since its absence results in an increase in neuronal excitability and enhanced excitotoxicity in vitro and in vivo. Furthermore, PrP(C) has been implicated in the inhibition of N-methyl-d-aspartic acid (NMDA)-mediated neurotransmission, and prion protein gene (Prnp) knockout mice show enhanced neuronal death in response to NMDA and kainate (KA). In this study, we demonstrate that neurotoxicity induced by KA in Prnp knockout mice depends on the c-Jun N-terminal kinase 3 (JNK3) pathway since Prnp(o/o)Jnk3(o/o) mice were not affected by KA. Pharmacological blockage of JNK3 activity impaired PrP(C)-dependent neurotoxicity. Furthermore, our results indicate that JNK3 activation depends on the interaction of PrP(C) with postsynaptic density 95 protein (PSD-95) and glutamate receptor 6/7 (GluR6/7). Indeed, GluR6-PSD-95 interaction after KA injections was favored by the absence of PrP(C). Finally, neurotoxicity in Prnp knockout mice was reversed by an AMPA/KA inhibitor (6,7-dinitroquinoxaline-2,3-dione) and the GluR6 antagonist NS-102. We conclude that the protection afforded by PrP(C) against KA is due to its ability to modulate GluR6/7-mediated neurotransmission and hence JNK3 activation.
    Tipo de documento:
    Referencia
    Referencia del producto:
    S7100
    Nombre del producto:
    ApopTag® Peroxidase In Situ Apoptosis Detection Kit

  • Oncogenicity of the developmental transcription factor Sox9. 22246670

    SOX9 [sex-determining region Y (SRY)-box 9 protein], a high mobility group box transcription factor, plays critical roles during embryogenesis and its activity is required for development, differentiation, and lineage commitment in various tissues including the intestinal epithelium. Here, we present functional and clinical data of a broadly important role for SOX9 in tumorigenesis. SOX9 was overexpressed in a wide range of human cancers, where its expression correlated with malignant character and progression. Gain of SOX9 copy number is detected in some primary colorectal cancers. SOX9 exhibited several pro-oncogenic properties, including the ability to promote proliferation, inhibit senescence, and collaborate with other oncogenes in neoplastic transformation. In primary mouse embryo fibroblasts and colorectal cancer cells, SOX9 expression facilitated tumor growth and progression whereas its inactivation reduced tumorigenicity. Mechanistically, we have found that Sox9 directly binds and activates the promoter of the polycomb Bmi1, whose upregulation represses the tumor suppressor Ink4a/Arf locus. In agreement with this, human colorectal cancers showed a positive correlation between expression levels of SOX9 and BMI1 and a negative correlation between SOX9 and ARF in clinical samples. Taken together, our findings provide direct mechanistic evidence of the involvement of SOX9 in neoplastic pathobiology, particularly, in colorectal cancer.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB5535
    Nombre del producto:
    Anti-Sox9 Antibody

  • Conditional deletion of the retinoblastoma (Rb) gene in ovarian granulosa cells leads to premature ovarian failure. 18599617

    The retinoblastoma protein (RB) regulates cell proliferation and survival by binding to the E2F family of transcription factors. Recent studies suggest that RB also regulates differentiation in a variety of cell types, including myocytes, neurons, adipocytes, and chondrocytes. Rb mutations have been found in ovarian cancer; however, the role of RB in normal and abnormal ovarian function remains unclear. To test the hypothesis that loss of Rb induces ovarian tumorigenesis, we generated an ovarian granulosa cell conditional knockout of Rb (Rb cKO) using the Cre/lox recombination system. Rb cKO females showed 100% survival and no ovarian tumor formation through 9 months of age, but they developed progressive infertility. Prepubertal Rb cKO females showed increased ovulation rates compared with controls, correlating with increased follicle recruitment, higher Fshr and Kitl mRNA levels, and lower anti-Müllerian hormone levels. In contrast, the ovulation rate of 6-wk-old females was similar to that of controls. Morphometric analysis of Rb cKO ovaries from 6-wk-old and older females showed increased follicular atresia and apoptosis. Rb cKO ovaries and preantral follicles had abnormal levels of known direct and indirect target genes of RB, including Rbl2/p130, E2f1, Ccne2, Myc, Fos, and Tgfb2. In addition, preantral follicles showed increased expression of the granulosa cell differentiation marker Inha, decreased levels of Foxl2 and Cyp19a1 aromatase, and abnormal expression of the nuclear receptors Nr5a1, Nr5a2, and Nr0b1. Taken together, our results suggest that RB is required for the temporal-specific pattern of expression of key genes involved in follicular development.
    Tipo de documento:
    Referencia
    Referencia del producto:
    S7111
    Nombre del producto:
    ApopTag® Plus In Situ Apoptosis Fluorescein Detection Kit

  • Rafs constitute a nodal point in the regulation of embryonic endothelial progenitor cell growth and differentiation. 18205709

    Mouse embryonic endothelial progenitor cells (eEPCs) acquire a mature phenotype after treatment with cyclic adenosine monophosphate (cAMP), suggesting an involvement of Raf serine/threonine kinases in the differentiation process. To test this idea, we investigated the role of B-Raf and C-Raf in proliferation and differentiation of eEPCs by expressing fusion proteins consisting of the kinase domains from Raf molecules and the hormone binding site of the estrogen receptor (ER), or its variant, the tamoxifen receptor. Our findings show that both B- and C-Raf kinase domains, when lacking adjacent regulatory parts, are equally effective in inducing eEPC differentiation. In contrast, the C-Raf kinase domain is a more potent stimulator of eEPC proliferation than B-Raf. In a complimentary approach, we used siRNA silencing to knockdown endogenously expressed B-Raf and C-Raf in eEPCs. In this experimental setting, we found that eEPCs lacking B-Raf failed to differentiate, whereas loss-of C-Raf function primarily slowed cell growth without impairing cAMP-induced differentiation. These findings were further corroborated in B-Raf null eEPCs, isolated from the corresponding knockout embryos, which failed to differentiate in vitro. Thus, gain- and loss-of-function experiments point to distinct roles of B-Raf and C-Raf in regulating growth and differentiation of endothelial progenitor cells, which may harbour therapeutic implications.
    Tipo de documento:
    Referencia
    Referencia del producto:
    07-453
    Nombre del producto:
    Anti-B-Raf Antibody

  • Assessment of caspase mediated degradation of linker for activation of T cells (LAT) at a single cell level. 23261919

    Caspase/Granzyme B mediated protein degradation is involved in elimination of activated T cell receptor (TCR) signaling molecules during processes of thymocyte selection and maintenance of peripheral homeostasis of T cells. Key components of TCR signaling cassette including LAT undergo biological inactivation in response to pro-apoptotic or anergy inducing environmental stimuli. Although available Western immunoblotting-based techniques are appropriate for detection of protein degradation in bulk populations of target cells, quantitative assessment of this process at a single cell level requires a different approach. Here we report on a novel, flow cytometry-based method for assessment of LAT integrity. This method exploits a loss of an anti-LAT antibody epitope recognition following proteolytic degradation of C-terminal domain of the LAT. We show that the LAT degradation precedes phosphatidylserine translocation to the outer leaflet of the plasma membrane and thus may constitute an early marker of T cell apoptosis. When used in conjunction with multi-parameter flow cytometry, our method revealed that FoxP3(+)CD4(+)CD8(low) thymocytes i.e. precursors of thymus derived CD4(+) regulatory T cells, in contrast to Foxp3(-)CD4(+)CD8(low) thymocytes are resistant to LAT degradation in response to CD3ε crosslinking. This finding can be used as an additional marker for T regulatory cell lineage.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-807
    Nombre del producto:
    Anti-LAT Antibody

  • Validation of real-time methylation-specific PCR to determine O6-methylguanine-DNA methyltransferase gene promoter methylation in glioma. 18556773

    Epigenetic silencing of the DNA repair protein O(6)-methylguanine-DNA methyltransferase (MGMT) by promoter methylation predicts successful alkylating agent therapy, such as with temozolomide, in glioblastoma patients. Stratified therapy assignment of patients in prospective clinical trials according to tumor MGMT status requires a standardized diagnostic test, suitable for high-throughput analysis of small amounts of formalin-fixed, paraffin-embedded tumor tissue. A direct, real-time methylation-specific PCR (MSP) assay was developed to determine methylation status of the MGMT gene promoter. Assay specificity was obtained by selective amplification of methylated DNA sequences of sodium bisulfite-modified DNA. The copy number of the methylated MGMT promoter, normalized to the beta-actin gene, provides a quantitative test result. We analyzed 134 clinical glioma samples, comparing the new test with the previously validated nested gel-based MSP assay, which yields a binary readout. A cut-off value for the MGMT methylation status was suggested by fitting a bimodal normal mixture model to the real-time results, supporting the hypothesis that there are two distinct populations within the test samples. Comparison of the tests showed high concordance of the results (82/91 [90%]; Cohen's kappa = 0.80; 95% confidence interval, 0.82-0.95). The direct, real-time MSP assay was highly reproducible (Pearson correlation 0.996) and showed valid test results for 93% (125/134) of samples compared with 75% (94/125) for the nested, gel-based MSP assay. This high-throughput test provides an important pharmacogenomic tool for individualized management of alkylating agent chemotherapy.
    Tipo de documento:
    Referencia
    Referencia del producto:
    S4530
    Nombre del producto:
    EX-WAX™ Paraffin-embedded DNA Extraction Kit

  • Protein kinase D1 mediates stimulation of DNA synthesis and proliferation in intestinal epithelial IEC-18 cells and in mouse intestinal crypts. 21051537

    We examined whether protein kinase D1 (PKD1), the founding member of a new protein kinase family, plays a critical role in intestinal epithelial cell proliferation. Our results demonstrate that PKD1 activation is sustained, whereas that of PKD2 is transient in intestinal epithelial IEC-18 stimulated with the G(q)-coupled receptor agonists angiotensin II or vasopressin. PKD1 gene silencing utilizing small interfering RNAs dramatically reduced DNA synthesis and cell proliferation in IEC-18 cells stimulated with G(q)-coupled receptor agonists. To clarify the role of PKD1 in intestinal epithelial cell proliferation in vivo, we generated transgenic mice that express elevated PKD1 protein in the intestinal epithelium. Transgenic PKD1 exhibited constitutive catalytic activity and phosphorylation at the activation loop residues Ser(744) and Ser(748) and on the autophosphorylation site, Ser(916). To examine whether PKD1 expression stimulates intestinal cell proliferation, we determined the rate of crypt cell DNA synthesis by detection of 5-bromo-2-deoxyuridine incorporated into the nuclei of crypt cells of the ileum. Our results demonstrate a significant increase (p less than 0.005) in DNA-synthesizing cells in the crypts of two independent lines of PKD1 transgenic mice as compared with non-transgenic littermates. Morphometric analysis showed a significant increase in the length and in the total number of cells per crypt in the transgenic PKD1 mice as compared with the non-transgenic littermates (p less than 0.01). Thus, transgenic PKD1 signaling increases the number of cells per crypt by stimulating the rate of crypt cell proliferation. Collectively, our results indicate that PKD1 plays a role in promoting cell proliferation in intestinal epithelial cells both in vitro and in vivo.
    Tipo de documento:
    Referencia
    Referencia del producto:
    04-787

  • Nuclear translation visualized by ribosome-bound nascent chain puromycylation. 22472439

    Whether protein translation occurs in the nucleus is contentious. To address this question, we developed the ribopuromycylation method (RPM), which visualizes translation in cells via standard immunofluorescence microscopy. The RPM is based on ribosome-catalyzed puromycylation of nascent chains immobilized on ribosomes by antibiotic chain elongation inhibitors followed by detection of puromycylated ribosome-bound nascent chains with a puromycin (PMY)-specific monoclonal antibody in fixed and permeabilized cells. The RPM correlates localized translation with myriad processes in cells and can be applied to any cell whose translation is sensitive to PMY. In this paper, we use the RPM to provide evidence for translation in the nucleoplasm and nucleolus, which is regulated by infectious and chemical stress.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MABE343
    Nombre del producto:
    Anti-Puromycin Antibody, clone 12D10

  • Identification of potential bladder cancer markers in urine by abundant-protein depletion coupled with quantitative proteomics. 23631828

    In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. Six apolipoproteins (APOA1, APOA2, APOB, APOC2, APOC3, and APOE) were able to differentiate bladder cancer from hernia. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity (AUC=0.80 and p<0.001) in discriminating bladder cancer from hernia than either marker alone. Using MetaCore software to interpret global changes of the urine proteome caused by bladder cancer, we found that the most notable alterations were in immune-response/alternative complement and blood-coagulation pathways. This study confirmed the clinical significance of the urine proteome in the development of non-invasive biomarkers for the detection of bladder cancer.In this study, we evaluated the reproducibility of abundant urine protein depletion by hexapeptide-based library beads and an antibody-based affinity column using the iTRAQ technique. The antibody-based affinity-depletion approach, which proved superior, was then applied in conjunction with iTRAQ to discover proteins that were differentially expressed between pooled urine samples from hernia and bladder cancer patients. Several proteins, including seven apolipoproteins, TIM, SAA4, and proEGF were further verified in 111 to 203 individual urine samples from patients with hernia, bladder cancer, or kidney cancer. SAA4 was significantly increased in bladder cancer subgroups, whereas ProEGF was significantly decreased in bladder cancer subgroups. Additionally, the combination of SAA4 and ProEGF exhibited higher diagnostic capacity in discriminating bladder cancer from hernia than either marker alone. A marker panel composed by two novel biomarker candidates, SAA4 and proEGF, was first discovered and verified successfully using Western blotting. To the best of our knowledge, the associations of urinary SAA4 and proEGF with bladder tumor and kidney cancer have not been mentioned before. In the present study, we discovered and verified SAA4 and proEGF as potential bladder cancer biomarker for the first time.
    Tipo de documento:
    Referencia
    Referencia del producto:
    APOMAG-62K
    Nombre del producto:
    MILLIPLEX MAP Human Apolipoprotein Magnetic Bead Panel - Cardiovascular Disease Multiplex Assay

  • Monoclonal antibody detection of CD46 clustering beneath Neisseria gonorrhoeae microcolonies 16552073

    CD46 (membrane cofactor protein), a complement-regulatory protein that participates in innate and acquired immunity, also serves as a receptor for viral and bacterial pathogens. CD46 isoforms terminate in one of two cytoplasmic tails, Cyt1 or Cyt2, which differ in signaling and trafficking properties. Dissecting the functions of the two cytoplasmic tails in these cellular processes has been hampered by the absence of specific reagents. Here we report the construction of Cyt1- and Cyt2-specific monoclonal antibodies (MAbs). These MAbs recognize unique epitopes within the tails and can be used for immunofluorescence microscopy, immunoblotting, and immunoprecipitation. Studies of Neisseria gonorrhoeae-infected cells with the CD46 tail MAbs demonstrate the differential recruitment of Cyt1 and Cyt2 to the cortical plaque.
    Tipo de documento:
    Referencia
    Referencia del producto:
    Múltiplo
    Nombre del producto:
    Múltiplo