DOCSLIB.ORG

Consequences of Loss of PINCH2 Expression in Mice

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Consequences of Loss of PINCH2 Expression in Mice Research Article 5899 Consequences of loss of PINCH2 expression in mice Fabio Stanchi1, Randi Bordoy1, Oliver Kudlaceck1, Attila Braun1, Alexander Pfeifer2, Markus Moser1 and Reinhard Fässler1,* 1Max Planck Institute of Biochemistry, Department of Molecular Medicine, 82152 Martinsried, Germany 2Department of Pharmacy, Center for Drug Research, University of Munich, 81377 Munich, Germany *Author for correspondence (e-mail: [email protected]) Accepted 12 September 2005 Journal of Cell Science 118, 5899-5910 Published by The Company of Biologists 2005 doi:10.1242/jcs.02686 Summary PINCH2 belongs, together with PINCH1, to a new family overlapping function in vivo. To further test this possibility, of focal adhesion proteins, the members of which are we established PINCH1-null mouse embryonic fibroblasts, composed of five LIM domains. PINCH1 and PINCH2 which express neither PINCH1 nor PINCH2. We found interact, through their first LIM domain, with the integrin- that in fibroblasts with a PINCH1/2-null background, linked kinase and thereby link integrins with several signal PINCH2 is able to rescue the spreading and adhesion transduction pathways. Despite their high similarity, it has defects of mutant fibroblasts to the same extent as PINCH1. been shown that PINCH1 and PINCH2 could exert distinct Furthermore, we show that the LIM1 domain only of either functions during cell spreading and cell survival. To PINCH1 or PINCH2 can prevent ILK degradation despite investigate the function of PINCH2 in vivo, we deleted their failure to localize to focal adhesions. Altogether these PINCH2 in mouse using the loxP/Cre system. In contrast results suggest that PINCH1 and PINCH2 share to the PINCH1-deficient mice, which die at the peri- overlapping functions and operate dependently and implantation stage, PINCH2-null mice are viable, fertile independently of their subcellular localization. and show no overt phenotype. Histological analysis of tissues that express high levels of PINCH2 such as bladder Supplementary material available online at and kidney revealed no apparent abnormalities, but http://jcs.biologists.org/cgi/content/full/118/24/5899/DC1 showed a significant upregulation of PINCH1, suggesting that the two PINCH proteins may have, at least in part, Key words: PINCH (Lims), ILK, Integrin, Adhesion, Migration Introduction function in vivo came from studies on UNC-97, the Journal of Cell Science Integrins are heterodimeric transmembrane glycoproteins that Caenorhabditis elegans orthologue of PINCH (Hobert et al., mediate cell-cell and cell-extracellular matrix adhesion 1999). Worms in which UNC-97 has been depleted by RNA (Hynes, 2002). They play pivotal roles in many biological interference arrest their development and show paralysis due processes including cell shape modulation, proliferation, to disrupted muscle attachment sites to the hypodermis. This survival and differentiation (van der Flier and Sonnenberg, phenotype resembles the defects observed in ␤ integrin- and 2001). Integrins execute these functions by recruiting a large ILK-null worms (Gettner et al., 1995; Mackinnon et al., 2002). number of intracellular adaptor and signaling molecules to Similarly, Drosophila melanogaster lacking the PINCH gene their short cytoplasmic domain (Zamir and Geiger, 2001). They suffers from muscle malfunction and paralysis caused by link integrins with different signaling pathways and connect impaired integrin-dependent cytoskeletal attachment to the integrin adhesion sites to the actin cytoskeleton. Integrin- plasma membrane (Clark et al., 2003). associated molecules can also modulate integrin activation Vertebrates have two PINCH isoforms, PINCH1 and (affinity) and their clustering in focal adhesion sites (FAs; PINCH2 (Braun et al., 2003; Zhang et al., 2002c). Both PINCH avidity). One FA protein is PINCH (also known as Lims), proteins share the same modular architecture and have high which is composed of five LIM domains followed by a short sequence similarity. The most noticeable difference between C-terminal tail containing putative nuclear localization/export the two PINCH proteins is the C-terminal tail, which is 11 signals. Each LIM domain is composed of two zinc-finger amino acids longer in PINCH2 than in PINCH1. In addition, motifs, which are able to engage in protein interactions the two PINCH genes share the same structure (Braun et al., (Kadrmas and Beckerle, 2004). The second zinc-finger of the 2003) suggesting that they have probably evolved by gene LIM1 domain of PINCH binds integrin-linked kinase (ILK) (Li duplication of a common ancestor, as have the members of et al., 1999). The LIM4 domain binds the adaptor protein many other vertebrate gene families (Gu et al., 2003). We NCK2, which interacts with several tyrosine kinase receptors previously compared the expression pattern of the two PINCH thereby linking integrins and growth factor signaling (Tu et al., genes during mouse development and in adult mouse tissues 1998). The LIM5 domain interacts with the Ras suppressor 1 (Braun et al., 2003). Interestingly, both in situ and northern blot (RSU1), which links PINCH to JNK signaling (Kadrmas et al., analysis indicated that only PINCH1 is expressed during early 2004; Dougherty et al., 2005). embryonic development, whereas PINCH2 expression is first The first evidence for an essential role of PINCH for integrin detectable in the second half of embryogenesis. Although in 5900 Journal of Cell Science 118 (24) adult tissues the PINCH1 and PINCH2 transcripts are detected selectively in the primitive endoderm of the implanting in almost all organs, the expression of the two isoforms can embryo (Li et al., 2005). diverge within different cell types of the same organ. For Whereas in vivo and in vitro studies provided insight into example, in colon PINCH2 expression is restricted to the the role of PINCH1, little is known about PINCH2. smooth muscle layer, whereas PINCH1 is also expressed in the Overexpression studies in HeLa cells revealed that PINCH2 epithelial layer (Braun et al., 2003). can compete with PINCH1 for ILK binding and hence alter the In order to analyze PINCH1 function in vivo we and others balance of PINCH1-ILK to PINCH2-ILK complexes (Zhang recently reported the phenotype of PINCH1-deficient mice, et al., 2002c). The appropriate balance seems to be essential which die shortly after implantation (Li et al., 2005; Liang et since re-expression of PINCH1 fully restored all the defects in al., 2005). Although loss of ␤1 integrin or ILK also leads to PINCH1 knock down cells, whereas expression of PINCH2 peri-implantation lethality (Fässler and Meyer, 1995; Stephens rescued ILK protein level, but not cell spreading and Akt et al., 1995; Sakai et al., 2003), PINCH1-null embryos survive phosphorylation (Fukuda et al., 2003; Xu et al., 2005). An significantly longer (Li et al., 2005). This prolonged additional functional difference between PINCH1 and development was confirmed in PINCH1-null embryoid bodies PINCH2 is the inability of PINCH2 to interact with the (EBs), which we generated from PINCH1-null ES cells. The PINCH1-binding protein RSU1 (Dougherty et al., 2005). All EB studies revealed that lack of PINCH1 permits these findings led to the proposal that PINCH2 may act as a differentiation of primitive endoderm and epiblast but their regulator of PINCH1 activity (Wu, 2004). adhesion to the basement membrane (BM), polarity, survival To investigate PINCH2 function in vivo, we generated and, surprisingly, cell-cell adhesion are severely compromised PINCH2 (Lims2 – Mouse Genome Informatics) knockout (Li et al., 2005). mice. To our surprise, these mice are viable, fertile and age In addition to our in vivo studies, many in vitro experiments normally. Tissues that normally express high levels of PINCH2 provided insight into the molecular functions of PINCH1. up-regulated PINCH1 expression. Complementation Overexpression of dominant negative PINCH1 protein or experiments with mouse embryonic fibroblasts (MEFs) that siRNA knock downs of PINCH1 in HeLa cells revealed an express neither PINCH1 nor PINCH2 revealed that PINCH2 is essential role in controlling cell spreading, adhesion and able to rescue their spreading and adhesion defects to the same migration (Fukuda et al., 2003; Zhang et al., 2002a). extent as PINCH1. Interestingly, the subcellular localization of Furthermore, these studies revealed that the interaction of either PINCH isoforms is essential to restore spreading and PINCH1 and ILK is required for the localization of a ternary adhesion but is not essential to prevent ILK degradation. complex composed of PINCH1, ILK and ␣-parvin to integrin adhesion sites as well as for the stability of each component of the complex. Depletion of PINCH1 leads to downregulation of Materials and Methods ILK protein and, vice versa, depletion of ILK leads to ES cells culture and generation of PINCH2-null mice downregulation of PINCH1 through proteasome-mediated A 500 bp fragment from a PINCH2 EST clone (GeneBank accession protein degradation (Fukuda et al., 2003). These findings are number AI325875) was used to screen a PAC library. Several clones in agreement with our PINCH1-null EBs, which also showed were identified and used to generate a conditional targeting PINCH2 Journal of Cell Science reduced ILK protein levels (Li et al., 2005). The molecular construct (PINCH2fl). Briefly, the targeting vector consists of a 4 kb left arm followed by a single loxP site, a 1.6 kb genomic fragment mechanism of how these proteins can mutually prevent their
Load more

Recommended publications
  • LIM Domain Proteins Pinch1/2 Regulate Chondrogenesis and Bone Mass in Mice
    Bone Research www.nature.com/boneres ARTICLE OPEN LIM domain proteins Pinch1/2 regulate chondrogenesis and bone mass in mice Yiming Lei1, Xuekun Fu1, Pengyu Li1, Sixiong Lin1,2, Qinnan Yan1, Yumei Lai3, Xin Liu1, Yishu Wang1, Xiaochun Bai 4, Chuanju Liu5,6, Di Chen7, Xuenong Zou2, Xu Cao8, Huiling Cao1 and Guozhi Xiao1 The LIM domain-containing proteins Pinch1/2 regulate integrin activation and cell–extracellular matrix interaction and adhesion. Here, we report that deleting Pinch1 in limb mesenchymal stem cells (MSCs) and Pinch2 globally (double knockout; dKO) in mice causes severe chondrodysplasia, while single mutant mice do not display marked defects. Pinch deletion decreases chondrocyte proliferation, accelerates cell differentiation and disrupts column formation. Pinch loss drastically reduces Smad2/3 protein expression in proliferative zone (PZ) chondrocytes and increases Runx2 and Col10a1 expression in both PZ and hypertrophic zone (HZ) chondrocytes. Pinch loss increases sclerostin and Rankl expression in HZ chondrocytes, reduces bone formation, and increases bone resorption, leading to low bone mass. In vitro studies revealed that Pinch1 and Smad2/3 colocalize in the nuclei of chondrocytes. Through its C-terminal region, Pinch1 interacts with Smad2/3 proteins. Pinch loss increases Smad2/3 ubiquitination and degradation in primary bone marrow stromal cells (BMSCs). Pinch loss reduces TGF-β-induced Smad2/3 phosphorylation and nuclear localization in primary BMSCs. Interestingly, compared to those from single mutant mice, BMSCs from dKO mice express dramatically lower protein levels of β-catenin and Yap1/Taz and display reduced osteogenic but increased adipogenic differentiation capacity. Finally, ablating Pinch1 in chondrocytes and Pinch2 globally causes severe osteopenia with subtle limb fi 1234567890();,: shortening.
    [Show full text]
  • Targeting of the EGFR/Β1 Integrin Connecting Proteins PINCH1 and Nck2 Radiosensitizes Three-Dimensional SCC Cell Cultures
    ONCOLOGY REPORTS 34: 469-476, 2015 Targeting of the EGFR/β1 integrin connecting proteins PINCH1 and Nck2 radiosensitizes three-dimensional SCC cell cultures LYDIA ROSSOW1,2, IRIS EKE1,2, ELLEN DICKREuTER1,2 and NILS CORDES1-5 1OncoRay-National Center for Radiation Research in Oncology, Faculty of Medicine and university Hospital Carl Gustav Carus, Technische universität Dresden, D-01307 Dresden, and Helmholtz-Zentrum Dresden-Rossendorf, D-01328 Dresden; 2Department of Radiation Oncology, university Hospital Carl Gustav Carus, Technische universität Dresden, Dresden; 3Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiooncology, Dresden; 4German Cancer Consortium (DKTK), D-01307 Dresden; 5German Cancer Research Center (DKFZ), D-69120 Heidelberg, Germany Received March 31, 2015; Accepted May 4, 2015 DOI: 10.3892/or.2015.4006 Abstract. Epidermal growth factor receptor (EGFR) signaling Introduction plays an important role in tumor cell resistance to therapy. In addition to ligand binding, mutual and cooperative interac- Epidermal growth factor receptor (EGFR) signaling is known tions of EGFR with integrin cell adhesion receptors critically to be deregulated in many human tumors (1,2). Causative are influence proper downstream signaling through a number of EGFR gene amplifications and mutations resulting in receptor bridging adapter proteins. In the present study, we analyzed overexpression and constitutively active EGFR tyrosine the role of two of these adapter proteins, called PINCH1 kinase activation. Due to its substantial role in
    [Show full text]
  • UNIVERSITY of CALIFORNIA RIVERSIDE Investigations Into The
    UNIVERSITY OF CALIFORNIA RIVERSIDE Investigations into the Role of TAF1-mediated Phosphorylation in Gene Regulation A Dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Cell, Molecular and Developmental Biology by Brian James Gadd December 2012 Dissertation Committee: Dr. Xuan Liu, Chairperson Dr. Frank Sauer Dr. Frances M. Sladek Copyright by Brian James Gadd 2012 The Dissertation of Brian James Gadd is approved Committee Chairperson University of California, Riverside Acknowledgments I am thankful to Dr. Liu for her patience and support over the last eight years. I am deeply indebted to my committee members, Dr. Frank Sauer and Dr. Frances Sladek for the insightful comments on my research and this dissertation. Thanks goes out to CMDB, especially Dr. Bachant, Dr. Springer and Kathy Redd for their support. Thanks to all the members of the Liu lab both past and present. A very special thanks to the members of the Sauer lab, including Silvia, Stephane, David, Matt, Stephen, Ninuo, Toby, Josh, Alice, Alex and Flora. You have made all the years here fly by and made them so enjoyable. From the Sladek lab I want to thank Eugene, John, Linh and Karthi. Special thanks go out to all the friends I’ve made over the years here. Chris, Amber, Stephane and David, thank you so much for feeding me, encouraging me and keeping me sane. Thanks to the brothers for all your encouragement and prayers. To any I haven’t mentioned by name, I promise I haven’t forgotten all you’ve done for me during my graduate years.
    [Show full text]
  • Anti-NCK2 Antibody (ARG59810)
    Product datasheet [email protected] ARG59810 Package: 100 μl anti-NCK2 antibody Store at: -20°C Summary Product Description Rabbit Polyclonal antibody recognizes NCK2 Tested Reactivity Hu, Ms, Rat Tested Application IHC-P, WB Host Rabbit Clonality Polyclonal Isotype IgG Target Name NCK2 Antigen Species Human Immunogen Recombinant fusion protein corresponding to aa. 1-380 of Human NCK2 (NP_003572.2). Conjugation Un-conjugated Alternate Names Nck-2; NCKbeta; NCK adaptor protein 2; SH2/SH3 adaptor protein NCK-beta; GRB4; Growth factor receptor-bound protein 4; Cytoplasmic protein NCK2 Application Instructions Application table Application Dilution IHC-P 1:50 - 1:200 WB 1:500 - 1:2000 Application Note * The dilutions indicate recommended starting dilutions and the optimal dilutions or concentrations should be determined by the scientist. Positive Control Rat brain, Mouse lung and 293T Calculated Mw 43 kDa Observed Size 43 kDa Properties Form Liquid Purification Affinity purified. Buffer PBS (pH 7.3), 0.02% Sodium azide and 50% Glycerol. Preservative 0.02% Sodium azide Stabilizer 50% Glycerol Storage instruction For continuous use, store undiluted antibody at 2-8°C for up to a week. For long-term storage, aliquot and store at -20°C. Storage in frost free freezers is not recommended. Avoid repeated freeze/thaw cycles. Suggest spin the vial prior to opening. The antibody solution should be gently mixed before use. www.arigobio.com 1/2 Note For laboratory research only, not for drug, diagnostic or other use. Bioinformation Gene Symbol NCK2 Gene Full Name NCK adaptor protein 2 Background This gene encodes a member of the NCK family of adaptor proteins.
    [Show full text]
  • Nck Adaptor Proteins Link Tks5 to Invadopodia Actin Regulation and ECM Degradation
    Research Article 2727 Nck adaptor proteins link Tks5 to invadopodia actin regulation and ECM degradation Stanley S. Stylli1, Stacey T. T. I1, Anne M. Verhagen2, San San Xu1, Ian Pass3, Sara A. Courtneidge3 and Peter Lock1,*,‡ 1Department of Surgery, University of Melbourne, Level 5, Clinical Sciences Building, Royal Melbourne Hospital, Parkville, Victoria 3052, Australia 2Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria 3052, Australia 3Burnham Institute for Medical Research, Torrey Pines Road, La Jolla, CA 92037, USA *Present address: Biochemistry Department, La Trobe University, Victoria 3086, Australia ‡Author for correspondence (e-mail: [email protected]) Accepted 5 May 2009 Journal of Cell Science 122, 2727-2740 Published by The Company of Biologists 2009 doi:10.1242/jcs.046680 Summary Invadopodia are actin-based projections enriched with overexpression and inhibited by Nck1 depletion. We show that proteases, which invasive cancer cells use to degrade the clustering at the plasma membrane of the Tks5 inter-SH3 region extracellular matrix (ECM). The Phox homology (PX)-Src containing Y557 triggers phosphorylation at this site, facilitating homology (SH)3 domain adaptor protein Tks5 (also known as Nck recruitment and F-actin assembly. These results identify a SH3PXD2A) cooperates with Src tyrosine kinase to promote Src-Tks5-Nck pathway in ECM-degrading invadopodia that invadopodia formation but the underlying pathway is not clear. shows parallels with pathways linking several mammalian and Here we show that Src phosphorylates Tks5 at Y557, inducing pathogen-derived proteins to local actin regulation. it to associate directly with the SH3-SH2 domain adaptor proteins Nck1 and Nck2 in invadopodia.
    [Show full text]
  • Thrombocytopenia-Associated Mutations in Ser/Thr Kinase MASTL
    Thrombocytopenia-associated mutations in Ser/Thr kinase MASTL deregulate actin cytoskeleton dynamics in platelets by Begoña Hurtado et al. SUPPLEMENTARY MATERIAL List of Supplementary Figures Figure S1. Hematopoietic precursors and megakaryocytes maturation in Mastl mutant mice. Figure S2. Sialylation and apoptosis profile of Mastl mutant platelets. Figure S3. Mastl E166D activity results in increased phosphorylation levels. Figure S4. Phospho-proteomic analysis in Mastl mutant platelets after thrombin activation. Figure S5. Signaling pathways differentially phosphorylated in Mastl mutant platelets. Figure S6. Changes in the phosphorylation status of signaling molecules in Mastl-mutant platelets. Figure S7. Full scan of blots showed in the manuscript. List of Supplementary Tables Table S1. Full data from phospho-proteomic studies. 1 Table S2. KEGG pathways enriched (FDR>0.05) in hyperphosphorylated proteins in resting Mastl(ED/ED) platelets (log2FC ED/WT>0.75), considering as statistical background the mouse platelet proteome. Table S3. KEGG pathways enriched (FDR>0.05) in hypophosphorylated proteins in resting Mastl(/) platelets (log2FC/WT<0.75), considering as statistical background the mouse platelet proteome. Table S4. KEGG pathways enriched (FDR>0.01) in hyperphosphorylated proteins in Mastl(ED/ED) platelets 3 minutes after stimulation with thrombin (log2FC ED/WT>0.75). Table S5. KEGG pathways enriched (FDR>0.01) in hyperphosphorylated proteins in both resting and 3-min-activated Mastl(ED/ED) platelets (log2FC ED/WT>0.5), considering as statistical background the mouse platelet proteome. Table S6. KEGG pathways enriched (FDR>0.05) in hyperphosphorylated proteins in Mastl(ED/ED) platelets 15 minutes after stimulation with thrombin (log2FC ED/WT>1.0), considering as statistical background the mouse platelet proteome.
    [Show full text]
  • Non-Overlapping Functions of Nck1 and Nck2 Adaptor Proteins in T Cell
    Ngoenkam et al. Cell Communication and Signaling 2014, 12:21 http://www.biosignaling.com/content/12/1/21 RESEARCH Open Access Non-overlapping functions of Nck1 and Nck2 adaptor proteins in T cell activation Jatuporn Ngoenkam1, Pussadee Paensuwan1, Kanlaya Preechanukul1, Boonruang Khamsri1, Ichaya Yiemwattana2, Esmeralda Beck-García3,4, Susana Minguet3, Wolfgang WA Schamel3 and Sutatip Pongcharoen5,6* Abstract Background: Signalling by the T cell antigen receptor (TCR) results in the activation of T lymphocytes. Nck1 and Nck2 are two highly related adaptor proteins downstream of the TCR that each contains three SH3 and one SH2 domains. Their individual functions and the roles of their SH3 domains in human T cells remain mostly unknown. Results: Using specific shRNA we down-regulated the expression of Nck1 or Nck2 to approximately 10% each in Jurkat T cells. We found that down-regulation of Nck1 impaired TCR-induced phosphorylation of the kinases Erk and MEK, activation of the AP-1 and NFAT transcription factors and subsequently, IL-2 and CD69 expression. In sharp contrast, down-regulation of Nck2 hardly impacts these activation read-outs. Thus, in contrast to Nck2, Nck1 is a positive regulator for TCR-induced stimulation of the Erk pathway. Mutation of the third SH3 domain of Nck1 showed that this domain was required for this activity. Further, TCR-induced NFAT activity was reduced in both Nck1 and Nck2 knock-down cells, showing that both isoforms are involved in NFAT activation. Lastly, we show that neither Nck isoform is upstream of p38 phosphorylation or Ca2+influx. Conclusions: In conclusion, Nck1 and Nck2 have non-redundant roles in human T cell activation in contrast to murine T cells.
    [Show full text]
  • Protein Context Shapes the Specificity of SH3 Domain-Mediated
    ARTICLE https://doi.org/10.1038/s41467-021-21873-2 OPEN Protein context shapes the specificity of SH3 domain-mediated interactions in vivo Ugo Dionne1,2,3,4,5, Émilie Bourgault3,4,5,6,13, Alexandre K. Dubé 3,4,5,6,7,13, David Bradley3,4,5,6,7,13, François J. M. Chartier 1,2,3,13, Rohan Dandage3,4,5,6,7, Soham Dibyachintan3,4,6,7,8, Philippe C. Després 3,4,5,6, Gerald D. Gish9, N. T. Hang Pham3,10, Myriam Létourneau3,10, ✉ ✉ Jean-Philippe Lambert 1,2,11, Nicolas Doucet3,10, Nicolas Bisson 1,2,3,12 & Christian R. Landry 3,4,5,6,7 1234567890():,; Protein–protein interactions (PPIs) between modular binding domains and their target pep- tide motifs are thought to largely depend on the intrinsic binding specificities of the domains. The large family of SRC Homology 3 (SH3) domains contribute to cellular processes via their ability to support such PPIs. While the intrinsic binding specificities of SH3 domains have been studied in vitro, whether each domain is necessary and sufficient to define PPI speci- ficity in vivo is largely unknown. Here, by combining deletion, mutation, swapping and shuffling of SH3 domains and measurements of their impact on protein interactions in yeast, we find that most SH3s do not dictate PPI specificity independently from their host protein in vivo. We show that the identity of the host protein and the position of the SH3 domains within their host are critical for PPI specificity, for cellular functions and for key biophysical processes such as phase separation.
    [Show full text]
  • Rpcmp: Robust P-Value Combination by Multiple Partitions with Applications to ATAC-Seq Data Menglan Cai and Limin Li*
    Cai and Li BMC Systems Biology 2018, 12(Suppl 9):141 https://doi.org/10.1186/s12918-018-0661-z RESEARCH Open Access rPCMP: robust p-value combination by multiple partitions with applications to ATAC-seq data Menglan Cai and Limin Li* From 29th International Conference on Genome Informatics Yunnan, China. 3-5 December 2018 Abstract Background: Evaluating the significance for a group of genes or proteins in a pathway or biological process for a disease could help researchers understand the mechanism of the disease. For example, identifying related pathways or gene functions for chromatin states of tumor-specific T cells will help determine whether T cells could reprogram or not, and further help design the cancer treatment strategy. Some existing p-value combination methods can be used in this scenario. However, these methods suffer from different disadvantages, and thus it is still challenging to design more powerful and robust statistical method. Results: The existing method of Group combined p-value (GCP) first partitions p-values to several groups using a set of several truncation points, but the method is often sensitive to these truncation points. Another method of adaptive rank truncated product method(ARTP) makes use of multiple truncation integers to adaptively combine the smallest p-values, but the method loses statistical power since it ignores the larger p-values. To tackle these problems, we propose a robust p-value combination method (rPCMP) by considering multiple partitions of p-values with different sets of truncation points. The proposed rPCMP statistic have a three-layer hierarchical structure. The inner-layer considers a statistic which combines p-values in a specified interval defined by two thresholds points, the intermediate-layer uses a GCP statistic which optimizes the statistic from the inner layer for a partition set of threshold points, and the outer-layer integrates the GCP statistic from multiple partitions of p-values.
    [Show full text]
  • CREB-Dependent Transcription in Astrocytes: Signalling Pathways, Gene Profiles and Neuroprotective Role in Brain Injury
    CREB-dependent transcription in astrocytes: signalling pathways, gene profiles and neuroprotective role in brain injury. Tesis doctoral Luis Pardo Fernández Bellaterra, Septiembre 2015 Instituto de Neurociencias Departamento de Bioquímica i Biologia Molecular Unidad de Bioquímica y Biologia Molecular Facultad de Medicina CREB-dependent transcription in astrocytes: signalling pathways, gene profiles and neuroprotective role in brain injury. Memoria del trabajo experimental para optar al grado de doctor, correspondiente al Programa de Doctorado en Neurociencias del Instituto de Neurociencias de la Universidad Autónoma de Barcelona, llevado a cabo por Luis Pardo Fernández bajo la dirección de la Dra. Elena Galea Rodríguez de Velasco y la Dra. Roser Masgrau Juanola, en el Instituto de Neurociencias de la Universidad Autónoma de Barcelona. Doctorando Directoras de tesis Luis Pardo Fernández Dra. Elena Galea Dra. Roser Masgrau In memoriam María Dolores Álvarez Durán Abuela, eres la culpable de que haya decidido recorrer el camino de la ciencia. Que estas líneas ayuden a conservar tu recuerdo. A mis padres y hermanos, A Meri INDEX I Summary 1 II Introduction 3 1 Astrocytes: physiology and pathology 5 1.1 Anatomical organization 6 1.2 Origins and heterogeneity 6 1.3 Astrocyte functions 8 1.3.1 Developmental functions 8 1.3.2 Neurovascular functions 9 1.3.3 Metabolic support 11 1.3.4 Homeostatic functions 13 1.3.5 Antioxidant functions 15 1.3.6 Signalling functions 15 1.4 Astrocytes in brain pathology 20 1.5 Reactive astrogliosis 22 2 The transcription
    [Show full text]
  • (NCK2) Rabbit Polyclonal Antibody – AP06442PU-N
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for AP06442PU-N Nck beta (NCK2) Rabbit Polyclonal Antibody Product data: Product Type: Primary Antibodies Applications: IHC Recommended Dilution: Immunohistochemistry on paraffin sections: 1/50-1/200. Reactivity: Human, Mouse Host: Rabbit Clonality: Polyclonal Immunogen: Synthetic peptide, corresponding to amino acids 341-390 of Human Nck-2. Specificity: This antibody detects endogenous levels of NCK2 protein. (region surrounding Ile364) Formulation: Phosphate buffered saline (PBS), pH 7.2. State: Aff - Purified State: Liquid purified Ig fraction Preservative: 0.05% sodium azide Concentration: 1.0 mg/ml Purification: Affinity-chromatography using epitope-specific immunogen and the purity is > 95% (by SDS- PAGE) Conjugation: Unconjugated Storage: Store undiluted at 2-8°C for one month or (in aliquots) at -20°C for longer. Avoid repeated freezing and thawing. Stability: Shelf life: one year from despatch. Predicted Protein Size: ~ 47 kDa Database Link: Entrez Gene 8440 Human O43639 This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 2 Nck beta (NCK2) Rabbit Polyclonal Antibody – AP06442PU-N Background: The NCK family of SH2/SH3 adaptor proteins consists of two members, NCK1 (NCKα) and NCK2 (NCKβ), which couple tyrosine kinase signaling, including the EGF and PDGF receptor- pathways, to downstream signaling proteins. Specifically, overexpression of NCK1 in NIH/3T3 cells decreases DNA synthesis stimulated by EGF.
    [Show full text]
  • Nck Beta (NCK2) Rabbit Polyclonal Antibody – TA354479 | Origene
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for TA354479 Nck beta (NCK2) Rabbit Polyclonal Antibody Product data: Product Type: Primary Antibodies Applications: IHC, WB Recommended Dilution: WB 0.1-1 µg/ml ELISA 0.01-0.1 µg/ml IP 2-5 µg/ml IHC 2-10 µg/ml FC 5-10 µg/ml Reactivity: Human Host: Rabbit Isotype: IgG Clonality: Polyclonal Immunogen: A synthetic peptide corresponding to the C-terminus of human NCK2 protein. Formulation: This affinity purified antibody is supplied in sterile Phosphate buffered saline (pH7.2) containing antibody stabilizer. Purification: The Rabbit IgG is purified by Epitope Affinity Purification Conjugation: Unconjugated Storage: Store at -20°C as received. Stability: Stable for 12 months from date of receipt. Predicted Protein Size: 45 kDa Gene Name: NCK adaptor protein 2 Database Link: NP_001004720 Entrez Gene 8440 Human O43639 Background: The NCK family of SH2/SH3 adaptor proteins consists of two members, NCK1 and NCK2, which couple tyrosine kinase signaling, including the EGF and PDGF receptor-pathways, to downstream signaling proteins. The NCK2 protein binds human PDGFR-b (Tyr-1009); overexpression of NCK2 inhibits PDGF-induced membrane ruffling and lamellipod formation. Synonyms: GRB4; NCKbeta Protein Families: Druggable Genome Protein Pathways: Axon guidance, ErbB signaling pathway, Pathogenic Escherichia coli infection, T cell receptor signaling pathway This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 2 Nck beta (NCK2) Rabbit Polyclonal Antibody – TA354479 Product images: WB: The cell lysate derived from MCF-7 was immunoblotted by the Rabbit anti-NCK2 antibody at 1:500.
    [Show full text]