Role of EBAG9 in COPI-dependent glycoprotein maturation and secretion processes in tumor cells [Elektronische Ressource] / von Jana Wolf, geb. Göttert

Jana Wolf

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2010
• Nombre de lectures: 14
• Langue: Deutsch
• Poids de l'ouvrage: 14 Mo

Extrait

Role of EBAG9 in COPI-Dependent
Glycoprotein Maturation and Secretion
Processes in Tumor Cells
Dissertation
zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)
im Fach Biologie
eingereicht an der

Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin
von
Diplom-Biologin Jana Wolf, geb. Göttert

Präsident der Humboldt-Universität zu Berlin
Prof. Dr. Dr. h.c. Christoph Markschies

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Dr. Andreas Herrmann

Gutachter/innen: 1. Prof. Dr. Thomas Börner
2. Prof. Dr. med. Bernd Dörken
3. PD Dr. Uta Höpken
Tag der mündlichen Prüfung: 21.10.2010


Index of contents

Index of contents
ABSTRACT......................................................................................................................................................... VI
ZUSAMMENFASSUNG....................................................................................................................................VII
1 INTRODUCTION......................................................................................................................................... 1
1.1 THE TUMOR ASSOCIATED ANTIGEN EBAG9 ............................................................................................. 1
1.1.1 Features of EBAG9 protein............................................................................................................. 1
1.1.2 EBAG9 and cancer.......................................................................................................................... 2
1.1.3 Biological functions of EBAG9 ...................................................................................................... 4
1.2 THE SECRETORY PATHWAY ....................................................................................................................... 5
1.2.1 From the endoplasmic reticulum to the cell exterior....................................................................... 5
1.2.1.1 Transport from the trans-Golgi-network............................................................................................ 5
1.2.1.2 Polarized transport ......................................................................................................................... 7
1.2.2 Mechanisms of biosynthetic protein transport from the ER towards the Golgi .............................. 9
1.2.3 From budding to fusion: components of the secretory pathway.................................................... 10
1.2.3.1 Coat proteins ............................................................................................................................... 10
1.2.3.2 The vesicle machinery: from Arfs to SNAREs................................................................................. 12
1.2.4 Retrograde transport...................................................................................................................... 15
1.3 BIOSYNTHETIC TRANSPORT AND GLYCOSYLATION ................................................................................. 17
1.3.1 N- and O-glycosylation ................................................................................................................. 17
1.3.2 Functional role of glycosylation.................................................................................................... 19
1.4 THE SECRETORY PATHWAY AND DISEASE ................................................................................................ 20
1.5 AIMS...................................................................................................................................................... 22
2 MATERIALS............................................................................................................................................... 24
2.1 CELLS .................................................................................................................................................... 24
2.2 VIRUSES................................................................................................................................................. 24
2.3 BACTERIAL STRAINS .............................................................................................................................. 25
2.4 MICE STRAINS........................................................................................................................................ 25
2.5 EXPRESSION CONSTRUCTS ..................................................................................................................... 25
2.6 OLIGONUCLEOTIDES .............................................................................................................................. 27
2.7 ANTIBODIES........................................................................................................................................... 27
2.8 REAGENTS ............................................................................................................................................. 30
2.9 BUFFER AND MEDIA ............................................................................................................................... 30
3 METHODS .................................................................................................................................................. 33
3.1 MOLECULAR BIOLOGY........................................................................................................................... 33
ii Index of contents

3.1.1 Culture of bacteria......................................................................................................................... 33
3.1.2 Transformation of E.coli ............................................................................................................... 33
3.1.3 Isolation of plasmid DNA from E.coli cells .................................................................................. 33
3.1.4 Polymerase chain reaction (PCR).................................................................................................. 34
3.1.5 Restriction digest........................................................................................................................... 34
3.1.6 Agarose gel electrophoresis .......................................................................................................... 34
3.1.7 Isolation of DNA from agarose gels.............................................................................................. 35
3.1.8 Ligation of DNA ........................................................................................................................... 35
3.1.9 Cloning.......................................................................................................................................... 35
3.2 PROTEIN BIOCHEMISTRY ........................................................................................................................ 35
3.2.1 Protein isolation and determination of protein concentration........................................................ 35
3.2.2 SDS-polyacrylamide-gelelectrophoresis (SDS-PAGE)................................................................. 36
3.2.3 Immunoblot (westernblot analysis)............................................................................................... 36
3.2.4 One-dimensional (1d)-isoelectric focusing gel electrophoresis (IEF)........................................... 37
3.2.5 Autoradiography ........................................................................................................................... 37
3.2.6 Quantification of protein bands..................................................................................................... 38
3.3 CELL BIOLOGY ....................................................................................................................................... 38
3.3.1 Cell culture and transfections........................................................................................................ 38
3.3.2 Transfection of mammalian cells .................................................................................................. 38
3.3.3 Generation of stable cell lines ....................................................................................................... 39
3.3.4 Amplification and transduction of cell lines with adenovirus....................................................... 39
3.3.5 Pulse-chase experiments ............................................................................................................... 40
3.3.6 Immunoprecipitation (IP) and co-immunoprecipitation................................................................ 40
3.3.7 Subcellular fractionation ............................................................................................................... 41
3.3.8 Fluorescence activated cell sorting (FACS)-analysis .................................................................... 42
3.3.9 Immunofluorescence ..................................................................................................................... 42
3.3.10 Lectin staining............................................................................................................................