Development and application of a high throughput cell based assay to identify novel modulators of ERK1/2 activation and, functional characterisation of the candidate radial spokehead like (Rshl1) [Elektronische Ressource] / presented by Meher Vinay Krishna Mohan Majety

Majety

Sujets

• Biologie

Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2006
• Nombre de lectures: 15
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences
















presented by
Meher Vinay Krishna Mohan Majety, M.Sc. in Biotechnology
Born in Vijayawada, India



Oral-examination:









Development and application of a high throughput cell based assay to
identify novel modulators of ERK1/2 activation and,
Functional characterisation of the candidate Radial spokehead like (Rshl1)

















Referees: PD. Dr. Stefan Wiemann
Prof. Dr. Ingrid Grummt




































To my Grandfather
Contents

SUMMARY............................................................................................................................... 1
ZUSAMENFASSUNG ............................................................................................................. 2
1 INTRODUCTION............................................................................................................ 3
1.1 The Mitogen Activated Protein Kinase pathway ................................................................................. 3
1.1.1 The Extra-cellular signal regulated Kinase (ERK) pathway and its mediators.................................... 4
1.1.2 Cytosolic substrates ............................................................................................................................. 6
1.1.3 Nuclear Targets of ERK1/2.................................................................................................................. 7
1.2 Regulation of ERK1/2 pathway ............................................................................................................. 8
1.2.1 Regulation via stimulus intensity and duration... 8
1.2.2 Regulation by Raf specificity............................................................................................................... 8
1.2.3 Regulation by cellular localisation....................................................................................................... 9
1.2.4 Regulation by scaffolding proteins ...................................................................................................... 9
1.2.5 Regulation by phosphatases ................................................................................................................. 9
1.2.6 Regulation by feed back inhibition....... 11
1.2.7 Cross talk between signalling pathways............................................................................................. 11
1.3 Physiological roles of ERK1/2 cascade................................................................................................ 11
1.3.1 Proliferation and Cell cycle................................................................................................................ 11
1.3.2 Differentiation.................................................................................................................................... 13
1.3.3 Apoptosis ........................................................................................................................................... 13
1.3.4 Cell Adhesion and Migration............................................................................................................. 13
1.4 ERK pathway and disease.................................................................................................................... 13
1.4.1 Cancer ................................................................................................................................................ 13
1.4.2 ERK pathway and cardiovascular diseases ........................................................................................ 14
1.4.3 Neuronal disorders............................................................................................................................. 14
1.5 Overview of the project................ 14
1.5.1 Detection of perturbations in ERK1/2 activity................................................................................... 16
1.7 High throughput cell based assay for identification of novel modulators in MAPK signalling..... 18
2 MATERIALS AND METHODS................................................................................... 19
2.1 Materials................................................................................................................................................ 19
2.1.1 Instruments and Equipment ............................................................................................................... 19
2.1.2 Plastics and Glassware....................................................................................................................... 20
2.1.3 Chemicals, Reagents and Media ........................................................................................................ 20
2.1.4 Kits..................................................................................................................................................... 22
2.1.5 Antibodies.......................................................................................................................................... 23
2.1.6 Peptides used for antibody generation ............................................................................................... 24
2.1.7 Buffers and Media.............................................................................................................................. 24
2.1.8 Antibiotics.......................................................................................................................................... 27
2.1.9 Restriction enzymes ........................................................................................................................... 27
2.1.10 Bacterial Strains.............. 27
2.1.11 Vectors .......................................................................................................................................... 28
2.1.11.1 Gateway entry vector................................................................................................................ 28
2.1.12 Cell lines.................. 29
2.2 Methods......................... 30
2.2.1 Polymerase Chain Reaction (PCR) .................................................................................................... 30
2.2.1.1 Generation of Entry clones....................................................................................................... 30
2.2.1.1.1 BP reaction ............................................................................................................................... 32
Contents

2.2.1.1.2 LR reaction............................................................................................................................... 33
2.2.2 Preparation of electro competent cells ............................................................................................... 34
2.2.3 Transformation of bacteria by electroporation................................................................................... 34
2.2.4 Isolation of plasmid DNA (Mini-prep) .............................................................................................. 35
2.2.5 Large scale preparation of plasmid DNA (Maxi prep)....................................................................... 35
2.2.6 Measuring the concentration of DNA ................................................................................................ 36
2.2.7 Restriction digest ............................................................................................................................... 36
2.2.8 Agarose gel electrophoresis ............................................................................................................... 37
2.2.9 Cell culture......................................................................................................................................... 37
2.2.9.1 Sub-culturing and maintenance of mammalian cells................................................................ 37
2.2.1.1 Cell counting using a Neuberger chamber................................................................................ 37
2.2.1.2 Transfection of mammalian cells 38
2.2.10 ß-galactosidase assay..................................................................................................................... 39
2.2.11 Protein extraction from mammalian cells...................................................................................... 39
2.2.12 n quantification .................................................................................................................... 40
2.2.12.1 Measurement of protein concentration at UV 280.................................................................... 40
2.2.12.2 Estimation of protein concentration using BCA (Bici