Investigations of intracellular EphB4 receptor tyrosine kinase signal transduction:
Literaturverz. S. 164 - 176. - Zsfassung in dt. Sprache.
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2008
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Zusammenfassung: | Literaturverz. S. 164 - 176. - Zsfassung in dt. Sprache. |
| Beschreibung: | V, 180 S. Ill., graph. Darst. |
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Datensatz im Suchindex
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| adam_text | Titel: Investigations of intracellular EphB4 receptor tyrosine kinase signal transduction
Autor: Rüther, Kathrin
Jahr: 2008
Content
Content
1 Introduction 1
1.1 The Eph family of receptor tyrosine kinases 1
1.1.1 Eph receptors and their ligands, the ephrins 1
; 1.1.2 Function and expression of Eph receptors 3
1.2 The role of EphB4 and ephrinB2 in vasculogenesis and
angiogenesis 4
1.3 Expression and function of Eph receptors and ephrin
ligands during tumor growth and tumor progression 6
1.4 Tumor-Angiogenesis 8
1.4.1 A historical view 8
1.4.2 Angiogenesis as a therapeutic target 9
1.4.3 EphB4 as a target for tumor anti-angiogenesis therapy . 11
1.5 Eph receptor signaling 12
1.5.1 Eph receptor activation 12
1.5.2 Eph receptor forward signaling 15
1.6 Aim of the work 18
2 Material and Methods 19
2.1 Material 19
2.1.1 Devices 19
2.1.2 Expendable items 21
2.1.2.1 Pipette tips 21
2.1.2.2 Reaction tubes 21
2.1.2.3 Cell cultures flasks and 96well plates 22
2.1.2.4 Others 22
2.1.3 Chemicals and biochemicals 23
2.1.3.1 Solids 23
2.1.3.2 Organic solvents 24
2.1.3.3 Liquids 24
2.1.3.4 Enzyme inhibitors 25
2.1.3.5 Gel electrophoresis and Western Blot
equipment 25
2.1.3.6 Immunoprecipitation and affinity
chromatography 26
2.1.3.7 Enzymes 27
I
Content
2.1.4 Kits 27
2.1.5 Antibodies 28
2.1.5.1 Primary antibodies 28
2.1.5.2 Secondary antibodies 29
2.1.6 Ready-to-use purchased buffers and solutions 29
2.2 Methods 30
2.2.1 Cell culture and preparation of cell lysates 30
2.2.1.1 Cell culture conditions 30
2.2.1.2 Cell lines and media and supplements 30
2.2.1.2.1 Cell lines 30
2.2.1.2.2 Media 31
2.2.1.2.3 Cell culture media supplements 32
2.2.1.2.4 Media compositions 33
2.2.1.3 Treatment of cells with ephrinB2-Fc 33
2.2 A A Preparation of total cell lysate with Duschl
lysis buffer 34
2.2.2 Protein analysis 35
2.2.2.1 Determination of protein concentration using
the Bio-RAD protein assay (Bradford s reagent) 35
2.2.2.2 ELISA to measure Y-phosphorylation
intensity of EphB4 35
2.2.2.3 FACS analysis 36
2.2.2.4 SDS-PAGE 37
2.2.2.4.1 Coomassie staining 38
2.2.2.4.2 Silver staining 38
2.2.2.5 Western blotting 39
2.2.2.5.1 Protein transfer on nitrocellulose
membranes 39
2.2.2.5.2 Detection of proteins with
specific antibodies 40
2.2.2.5.3 Removal of antibodies from !
nitrocellulose membranes 41
2.2.2.6 2-D gel electrophoresis 41
2.2.2.6.1 Preparation of proteins for 2-D
gel electrophoresis 41
2.2.2.6.2 First dimension: Isoelectric
focusing (IEF) 42
2.2.2.6.3 Second dimension: SDS-PAGE 43
2.2.2.7 Masspectrometry 43
2.2.2.8 Hypomatrix antibody array 46
II
Content
2.2.2.9 Prolifration assay 47
2.2.2.10 Beadlyte® 8-plex Multi-Pathway
Signaling assay 47
2.2.3 Protein purification 49
2.2.3.1 Immunoprecipitation 49
2.2.3.2 Purification of his-tagged EphB4 with
Ni-NTA-beads (IMAC) 49
2.2.3.3 Generation of affinity beads using CnBr
activated sepharose 51
2.2.3.4 Immunoaffinity purification of EphB4 52
2.2.4 Generation of polyclonal antibodies against EphB4 54
2.2 A A Expression and purification of a recombinant
GST-EphB4-SAM domain fusion protein 54
2.2.4.2 Generation of an aEphB4-SAM serum 54
2.2.4.3 Affinity purification of EphB4 specific
antibodies 55
2.2.4.3.1 Purification procedure 55
2.2.4.3.2 The aEphB4-SAM domain antibody
titer is measured with an ELISA 55
2.2.4.3.3 ELISA to determine the titer
contaminating aGST IgG 56
2.2.5 mRNA analysis 56
2.2.5.1 Preparation of mRNA from cultured
eukaryontic cells 56
2.2.5.2 RT-PCR analysis 57
2.2.5.2.1 First strand synthesis 57
2.2.5.2.2 PCR 58
2.2.5.3 DNA agaorse gel electrophoresis 59
3 Results 61
3.1 Investigation of EphB4 signal transduction in
CHO-EphB4 cells 61
3.1.1 Introduction of the CHO-EphB4mychis surrogate
system 61
3.1.1.1 Expression analysis of EphB receptors and
ephrinB2 in CHO and CHO-EphB4 cells 61
3.1.1.2 Analysis of EphB4 expression and its
membrane localization in CHO-EphB4 cells
by FACS 62
3.1.1.3 EphB4 receptor activation induces
Y-phosphorylation of intracellular proteins
III
Content
in CHO-EphB4 cells 63
3.1.2 EphB4 receptor phosphorylation kinetics in
CHO-EphB4 cells 64
3.1.3 EphB4 could be precipitated using ac-myc
protein A beads 66
3.1.4 EphB4 candidate binding proteins were not identified
in co-immunoprecipitation experiments and Western
blotting 67
3.1.5 Isolation of EphB4 receptor from CHO-EphB4 cells
using Ni-NTA chromatography 69
3.1.6 2-D gel electrophoresis of proteins isolated from
CHO-EphB4 cells with Ni-NTA chromatography 71
3.1.7 Purification of EphB4 from CHO-EphB4 cell lysates
using a stable ac-myc immunoaffinity resin 75
3.1.8 Identification of Carm-1 and hnRNP U in eluates of
CHO-EphB4 from the ac-myc immunoresin 78
3.1.9 Carm-1 does not specifically interact with EphB4 83
3.1.9.1 Identification of Carm-1 in eluates of
CHO-EphB4 and CHO control from the
ac-myc immunoresin 83
3.1.9.2 EphB4 does not co-precipitate with Carm-1 84
3.1.10 pY-EphB4 does selectively co-precipitate with
hnRNP U 85
3.2 Expression analysis of EphB4 in human tumor
and endothelial cells 88
3.2.1 Human tumor and endothelial cells express
mRNA for EphB receptors and the ligand ephrinB2 88
3.2.2 Expression of EphB4 protein in human tumor cell
lines and endothelial cells 91
3.2.3 Generation of antibodies directed against the
EphB4-SAM domain 92
3.2.4 Analysis of EphB4 activation in human tumor
cell lines and endothelial cells 95
3.2.5 Analysis of the cell surface localization of EphB4
and ephrinB2 by FACS 97
3.3 Analysis of intracellular EphB4 signal transduction in the
mammary breast tumor cell line MCF 7 99
IV
Content
3.3.1 EphB4 receptor phosphorylation kinetics in
MCF 7 cells 99
3.3.2 ephrinB2-Fc shows no effect on cell proliferation
of MCF 7 101
3.3.3 EphB4 receptor activation induces
Y-phosphorylation of proteins in MCF 7 cells 102
3.3.4 EphB4 does not signal via well known intracellular
pathways 104
3.3.5 The Hypomatrix antibody array does not reveal
EphB4 mediated changes in Y-phosphorylation
of specific proteins 105
3.3.6 EphB4 activation does not affect RasGAP
Y-phosphorylation in MCF 7 cells 107
3.3.7 Isolation of EphB4 from MCF 7 cell lysates by
affinity chromatography 109
3.3.8 hnRNP K does not co-immunoprecipitate with EphB4 . 115
4 Discussion 116
4.1 The CHO-EphB4 surrogate system 117
4.2 EphB4 expression in human tumor cell lines 128
4.3 EphB4 signal transduction in MCF 7 cells 133
4.4 Identification of EphB4 protein complexes with
immunoaffinity chromatography and mass spectrometry 142
4.5 Conclusion 147
4.6 Future perspectives 148
5 Summary 149
6 Zusammenfassung 150
7 Abbreviation Index 151
8 List of Figures 154
9 List of Tables 156
10 Supplements 158
11 Literature Index 164
12 Acknowledgment 177
13 Curriculum Vitae 179
V
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| spelling | Rüther, Kathrin 1976- Verfasser (DE-588)136997260 aut Investigations of intracellular EphB4 receptor tyrosine kinase signal transduction von Kathrin Rüther 2008 V, 180 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Berlin, Univ., Diss., 2008 Literaturverz. S. 164 - 176. - Zsfassung in dt. Sprache. Signaltransduktion (DE-588)4318717-1 gnd rswk-swf Protein-Tyrosin-Kinasen (DE-588)4409305-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Protein-Tyrosin-Kinasen (DE-588)4409305-6 s Signaltransduktion (DE-588)4318717-1 s DE-604 HBZ Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=017056144&sequence=000002&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Rüther, Kathrin 1976- Investigations of intracellular EphB4 receptor tyrosine kinase signal transduction Signaltransduktion (DE-588)4318717-1 gnd Protein-Tyrosin-Kinasen (DE-588)4409305-6 gnd |
| subject_GND | (DE-588)4318717-1 (DE-588)4409305-6 (DE-588)4113937-9 |
| title | Investigations of intracellular EphB4 receptor tyrosine kinase signal transduction |
| title_auth | Investigations of intracellular EphB4 receptor tyrosine kinase signal transduction |
| title_exact_search | Investigations of intracellular EphB4 receptor tyrosine kinase signal transduction |
| title_full | Investigations of intracellular EphB4 receptor tyrosine kinase signal transduction von Kathrin Rüther |
| title_fullStr | Investigations of intracellular EphB4 receptor tyrosine kinase signal transduction von Kathrin Rüther |
| title_full_unstemmed | Investigations of intracellular EphB4 receptor tyrosine kinase signal transduction von Kathrin Rüther |
| title_short | Investigations of intracellular EphB4 receptor tyrosine kinase signal transduction |
| title_sort | investigations of intracellular ephb4 receptor tyrosine kinase signal transduction |
| topic | Signaltransduktion (DE-588)4318717-1 gnd Protein-Tyrosin-Kinasen (DE-588)4409305-6 gnd |
| topic_facet | Signaltransduktion Protein-Tyrosin-Kinasen Hochschulschrift |
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