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  • Association of adaptor protein TRIP8b with clathrin. 21749376

    TPR-containing Rab8b-interacting protein (TRIP8b) is a brain-specific hydrophilic cytosolic protein that contains tetratricopeptide repeats (TPRs). Previous studies revealed interaction of this protein via its TPR-containing domain with Rab8b small GTPase, hyperpolarization-activated cyclic nucleotide-regulated channel (HCN) channels and G protein-coupled receptor calcium-independent receptor of α-latrotoxin. We identified clathrin as a major component of eluates from the TRIP8b affinity matrix. In the present study, by in vitro-binding analysis we demonstrate a direct interaction between clathrin and TRIP8b. The clathrin-binding site was localized in the N-terminal (non-TPR containing) part of the TRIP8b molecule that contains two short motifs involved in the clathrin binding. In transfected HEK293 cells, co-expression of HCN1 with TRIP8b resulted in translocation of the channels from the cell surface to large intracellular puncta where both TRIP8b and clathrin were concentrated. These puncta co-localized partially with an early endosome marker and strongly overlapped with lysosome staining reagent. When HCN1 was co-expressed with a clathrin-non-binding mutant of TRIP8b, clathrin did not translocate to HCN1 and TRIP8b-containing puncta, suggesting that TRIP8b interacts with HCN and clathrin independently. We found TRIP8b present in the fraction of clathrin-coated vesicles purified from brain tissues. Stripping the clathrin coat proteins from the vesicles with Tris alkaline buffer resulted in concomitant release of TRIP8b. Our data suggest complex regulatory functions of TRIP8b in neuronal endocytosis through independent interaction with membrane proteins and components of the clathrin coat.
    Tipo de documento:
    Referencia
    Referencia del producto:
    CBL188
    Nombre del producto:
    Anti-Clathrin Antibody, clone CHC5.9

  • aPKC acts upstream of PAR-1b in both the establishment and maintenance of mammalian epithelial polarity. 15324659

    BACKGROUND: aPKC and PAR-1 are required for cell polarity in various contexts. In mammalian epithelial cells, aPKC localizes at tight junctions (TJs) and plays an indispensable role in the development of asymmetric intercellular junctions essential for the establishment and maintenance of apicobasal polarity. On the other hand, one of the mammalian PAR-1 kinases, PAR-1b/EMK1/MARK2, localizes to the lateral membrane in a complimentary manner with aPKC, but little is known about its role in apicobasal polarity of epithelial cells as well as its functional relationship with aPKC. RESULTS: We demonstrate that PAR-1b is essential for the asymmetric development of membrane domains of polarized MDCK cells. Nonetheless, it is not required for the junctional localization of aPKC nor the formation of TJs, suggesting that PAR-1b works downstream of aPKC during epithelial cell polarization. On the other hand, aPKC phosphorylates threonine 595 of PAR-1b and enhances its binding with 14-3-3/PAR-5. In polarized MDCK cells, T595 phosphorylation and 14-3-3 binding are observed only in the soluble form of PAR-1b, and okadaic acid treatment induces T595-dependent dissociation of PAR-1b from the lateral membrane. Furthermore, T595A mutation induces not only PAR-1b leakage into the apical membrane, but also abnormal development of membrane domains. These results suggest that in polarized epithelial cells, aPKC phosphorylates PAR-1b at TJs, and in cooperation with 14-3-3, promotes the dissociation of PAR-1b from the lateral membrane to regulate PAR-1b activity for the membrane domain development. CONCLUSIONS: These results suggest that mammalian aPKC functions upstream of PAR-1b in both the establishment and maintenance of epithelial cell polarity.
    Tipo de documento:
    Referencia
    Referencia del producto:
    3460

  • Protein cage nanoparticles bearing the LyP-1 peptide for enhanced imaging of macrophage-rich vascular lesions. 21391720

    Cage-like protein nanoparticles are promising platforms for cell- and tissue-specific targeted delivery of imaging and therapeutic agents. Here, we have successfully modified the 12 nm small heat shock protein from Methanococcus jannaschii (MjHsp) to detect atherosclerotic plaque lesions in a mouse model system. As macrophages are centrally involved in the initiation and progression of atherosclerosis, targeted imaging of macrophages is valuable to assess the biologic status of the blood vessel wall. LyP-1, a nine residue peptide, has been shown to target tumor-associated macrophages. Thus, LyP-1 was genetically incorporated onto the exterior surface of MjHsp, while a fluorescent molecule (Cy5.5) was conjugated on the interior cavity. This bioengineered protein cage, LyP-Hsp, exhibited enhanced affinity to macrophage in vitro. Furthermore, in vivo injection of LyP-Hsp allowed visualization of macrophage-rich murine carotid lesions by in situ and ex vivo fluorescence imaging. These results demonstrate the potential of LyP-1-conjugated protein cages as nanoscale platforms for delivery of imaging agents for the diagnosis of atherosclerosis.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1976F
    Nombre del producto:
    Anti-Integrin αVβ3 Antibody, clone LM609, FITC conjugated

  • C-propeptides of procollagens I alpha 1 and II that differentially accumulate in enchondromas versus chondrosarcomas regulate tumor cell survival and migration. 20460531

    Chondrogenic tumors that exhibit benign or malignant behaviors synthesize variable amounts of cartilage-like extracellular matrix. To define the regulators of these phenotypes, we performed a proteomic comparison of multiple human chondrogenic tumors, which revealed differential accumulation of the C-propeptides of procollagens Ialpha1 and II (PC1CP and PC2CP) in malignant versus benign tumors, respectively. Expression patterns of PC1CP correlated with levels of tumor vascularization, whereas expression patterns of PC2CP suggested its susceptibility to immobilization within the extracellular matrix. Prompted by these observations, we investigated the functions of recombinant PC1CP and PC2CP in the extracellular matrix in soluble or immobilized states. Each induced beta1 integrin-mediated chondrocyte adhesion by distinct domains and efficacies, suggesting that they initiated distinct signaling pathways. Indeed, immobilized PC2CP, but not PC1CP, induced apoptosis of primary chondrocytes and EAhy926 endothelial cells. In contrast, soluble PC1CP, but not PC2CP, induced the migration of EAhy926 cells and increased vascular endothelial growth factor (VEGF) and CXCR4 expression in chondrocytes. Soluble PC2CP also increased VEGF expression, but along with a more pronounced effect on CXCR4 and matrix metalloproteinase 13 expression. Our findings suggest that PC1CP favors angiogenesis and tumor progression, but that PC2CP acts in a more complex manner, exerting antitumor and antiangiogenic properties through apoptosis induction when immobilized, but progression and metastasis when soluble. In summary, the relative levels of PC1CP and PC2CP and their interactions within the extracellular matrix contribute to tumor progression, angiogenesis, and metastasis in chondrogenic tumors.
    Tipo de documento:
    Referencia
    Referencia del producto:
    MAB1959
    Nombre del producto:
    Anti-Integrin β1 Antibody, clone P5D2

  • Plk4 is required for cytokinesis and maintenance of chromosomal stability. 20348415

    Aneuploidy is a characteristic feature of established cancers and can promote tumor development. Aneuploidy may arise directly, through unequal distribution of chromosomes into daughter cells, or indirectly, through a tetraploid intermediate. The polo family kinase Plk4/Sak is required for late mitotic progression and is haploinsufficient for tumor suppression in mice. Here we show that loss of heterozygosity (LOH) occurs at the Plk4 locus in 50% of human hepatocellular carcinomas (HCC) and is present even in preneoplastic cirrhotic liver nodules. LOH at Plk4 is associated with reduced Plk4 expression in HCC tumors but not with mutations in the remaining allele. Plk4(+/-) murine embryonic fibroblasts (MEFs) at early passage show a high incidence of multinucleation, supernumerary centrosomes, and a near-tetraploid karyotype. Underlying these phenotypes is a high rate of primary cytokinesis failure, associated with aberrant actomyosin ring formation, reduced RhoA activation, and failure to localize the RhoA guanine nucleotide exchange factor Ect2 to the spindle midbody. We further show that Plk4 normally localizes to the midbody and binds to and phosphorylates Ect2 in vitro. With serial passaging Plk4(+/-) MEFs rapidly immortalize, acquiring an increasing burden of nonclonal and clonal gross chromosomal irregularities, and form tumors in vivo. Our results indicate that haploid levels of Plk4 disrupt RhoGTPase function during cytokinesis, resulting in aneuploidy and tumorigenesis, thus implicating early LOH at Plk4 as one of the drivers of human hepatocellular carcinogenesis. These findings represent an advance in our understanding of genetic predisposition to HCC, which continues to increase in incidence globally and particularly in North America.
    Tipo de documento:
    Referencia
    Referencia del producto:
    06-813
    Nombre del producto:
    Anti-PLK1 Antibody, NT

  • Calcineurin-independent inhibition of 3T3-L1 adipogenesis by Pasteurella multocida toxin: suppression of Notch1, stabilization of beta-catenin and pre-adipocyte factor 1. 17581254

    Pasteurella multocida toxin (PMT) is a potent mitogen and a specific activator of Gq-dependent signalling pathways. PMT impairs osteoblast differentiation and causes bone loss and fat reduction in vivo. We examined the effect of PMT on cell signalling pathways involved in 3T3-L1 adipocyte differentiation. We demonstrate that PMT treatment before or together with differentiation induction factors inhibits adipogenesis and prevents upregulation of important adipocyte markers - peroxisome-proliferator-activated receptor gamma (PPARgamma) and CAATT enhancer-binding protein alpha (C/EBPalpha). Moreover, PMT completely downregulates PPARgamma and C/EBPalpha expression in mature adipocytes. Differentiation of pre-adipocytes into adipocytes requires the suppression of pre-adipocyte factor 1 (Pref1) and Wnt signalling, along with the degradation of beta-catenin. PMT prevents downregulation of Pref1 and beta-catenin under differentiation-inducing conditions. In addition, PMT treatment downregulates expression of Notch1, a protein responsible for cell fate decision and implicated in regulation of adipogenesis in 3T3-L1 cells. PMT action on adipogenesis was not reversed by cyclosporin A, an inhibitor of Galphaq-PLC-calcium-dependent calcineurin activation. Our results reveal new pathways involved in PMT action on cellular physiology and differentiation. Our study further demonstrates that the effect of PMT on Pref1/PPARgamma/C/EBPalpha expression and adipogenesis does not occur just through activation of the Galphaq-calcium-calcineurin pathway, but involves Wnt/beta-catenin and Notch1 signalling pathways, two signalling pathways strongly linked to cancer predisposition, neurological and immunological dysfunctions, and fat and bone development.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB3511

  • Systematic survey of deubiquitinase localization identifies USP21 as a regulator of centrosome- and microtubule-associated functions. 22298430

    Ubiquitination is a reversible modification that influences a broad range of physiological processes. There are approximately 90 deubiquitinases (DUBs) encoded in the human genome, of which 79 are predicted to have catalytic activity. We tagged 66 DUBs with green fluorescent protein and systematically surveyed their subcellular distribution, identifying enzymes specific to the nucleus, plasma membrane, and secretory and endocytic pathways. USP21 is unique in showing clear association with both centrosomes and microtubules. Using an in vitro assay, we show that microtubule binding is direct and identify a novel microtubule-binding motif encompassed within amino acids 59-75 of the N-terminus of USP21. Our functional studies indicate a key role for USP21 in the governance of microtubule- and centrosome-associated physiological processes: Depletion of USP21 in A549 cells compromises the reestablishment of a radial array of microtubules during recovery from cold-induced depolymerization and also reduces the probability of primary cilium formation, whereas USP21 knockdown in PC12 cells inhibits nerve growth factor-induced neurite outgrowth.
    Tipo de documento:
    Referencia
    Referencia del producto:
    AB6046
    Nombre del producto:
    Anti-Cytoplasmic FMR1-interacting protein 1 Antibody

  • High-throughput fluorescence polarization method for identification of FKBP12 ligands. 14599350

    FKBP12 is best known as the target of the widely used immunosuppressive drug FK506 but may also play a role in neuronal survival. Nonimmunosuppressive ligands of FKBP12 have been shown to have neuroprotective and neuroregenerative activity both in vitro and in vivo, stimulating interest in the development of high-throughput screens to rapidly identify novel ligands. FKBP12 was expressed as a His(6)-fusion in bacteria and purified by metal ion affinity and gel filtration chromatography. A high-throughput fluorescence polarization assay was developed to identify novel ligands of FKBP12. Dissociation constant values of known FKBP12 ligands measured by the new method agreed closely with K(i) values obtained by assaying inhibition of the rotamase activity of the enzyme. The fluorescence polarization assay is rapid, robust, and inexpensive and does not generate radioactive waste. It is very well suited for high-throughput screening efforts.
    Tipo de documento:
    Referencia
    Referencia del producto:
    20-181

  • Identification of NDRG1 as an early inducible gene during in vitro maturation of cultured mast cells. 12804568

    Coculture of mouse bone marrow-derived mast cells (BMMC) with fibroblasts in the presence of stem cell factor (SCF) facilitates morphological and functional maturation toward a connective tissue mast cell (CTMC)-like phenotype. By means of cDNA subtraction, we identified several inducible genes during this mast cell maturation process. Of approximately 100 sequenced clones induced, nearly 50% were chromosome 14-associated serine proteases. Approximately 14% encoded NDRG1, a 43-kDa cytosolic protein that has been implicated in cell differentiation. NDRG1 was distributed in the cytosol of cultured mast cells and CTMC in rat skin. Overexpression of NDRG1 in RBL-2H3 cells resulted in enhanced degranulation in response to various stimuli. Thus, NDRG1 may be a mast cell maturation-associated inducible protein that allows the cells to be susceptible to extracellular stimuli leading to degranulation. Additionally, several unique maturation-associated inducible genes were identified, molecular and functional characterization of which will provide new insights into mast cell biology.
    Tipo de documento:
    Referencia
    Referencia del producto:
    ECM600
    Nombre del producto:
    uPA Activity Assay Kit

  • Maternally transmitted partial direct tandem duplication of mitochondrial DNA associated with diabetes mellitus. 8268914

    Mitochondrial DNA from a 38 year old male with diabetes mellitus and features of mitochondrial dysfunction was analysed and shown to include a population with a partial duplication. The partially duplicated mitochondrial DNA molecules were evident in both muscle and blood. The region of mitochondrial DNA duplicated includes the origin of heavy strand replication, but not the light strand origin. This patient has features in common with other cases of partial direct tandem duplications and with a family which was reported to harbour a 10.4 kb mtDNA deletion. Initial restriction enzyme analysis of our case produced results consistent with a partial deletion of mitochondrial DNA. This leads us to propose that the rarity of reports of partial mitochondrial DNA duplications may stem in part from the classification of such mutants as partial deletions.
    Tipo de documento:
    Referencia
    Referencia del producto:
    12-408
    Nombre del producto:
    Histone H2B Peptide, biotin conjugate, residues 21-41