Antibody array for the detection of protein phosphorylation & localization and application to dissect activities of different signaling pathways [Elektronische Ressource] / von Lin Wang

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Sujets

• Biologie

Informations

• Publié par: friedrich-schiller-universitat_jena
• Publié le: 01 janvier 2004
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

Antibody Array for the Detection of Protein
Phosphorylation & Localization and Application to Dissect
Activities of Different Signaling Pathways








Dissertation

Zur Erlangung des akademischen Grades
doctor rerum naturalium
(Dr. rer. nat.)

Vorgelegt dem Rat der Biologisch-Pharmazeutischen Fakultät
der Friedrich-Schiller-Universität Jena





Von
Lin Wang
from Jinan born March12,1964











TABLES OF CONTENTS

1 Introduction................................................................................................................................... 1

1.1 Antibody Array Technologies............................................................................................. 1

1.2 Protein Phosphorylation, Localization and Function....................................................... 2

1.3 Interaction Proteomics and Pathway Building.................................................................... 3

1.3.1 BMP2 Signalling ............................................................................................................. 4
1.3.2 The Effect of STI 571 on Cell Signalling ........................................................................ 9
1.4 Aim ........................................................................................................................................... 10
2 Materials and Methods................................................................................................................. 11

2.1 Materials............................................................................................................................... 11
2.1.1 Cell Lines.................................................................................................................... 11
2.1.2 Chemicals................................................................................................................... 11
2.1.3 Antibodies................................................................................................................... 11
2.1.4 Protein-Molecular Weight Marker............................................................................... 11
2.1.5 Apparatus................................................................................................................... 11

2.2 Methods................................................................................................................................... 12
2.2.1 Cell Culture....................................................................................................................... 12
2.2.2 Cell Numbers and Experimental Format........................................................................... 12
2.2.3 Cytoplasmic and Nuclear Protein Extracts........................................................ 12
2.2.4 Determination of Protein Concentration............................................................................ 13
2.2.5 Ponceau S Staining ......................................................................................................... 13
2.2.6 Western Analysis ............................................................................................................. 14
2.2.7 Immunoprecipitation......................................................................................................... 14
2.2.8 Cy3 and Cy5 Staining in SDS-PAGE................................ 14
2.2.9 Cy3and Cy5 Staining of Microscope-Slide-Size Array......................................................14
2.2.10 Cy3 and Cy5 Staining of Mini-Arrayslide....................................................................... 15
1 Silver Staining of Mini-Arrayslide................................................................................... 15
2.2.12 Silver Staining in Arraytube........................................................................................... 15
2.2.12.1 Biotinylation of Cellular Proteins......................................................................... 15
2.2.12.2 Incubation of Protein Extracts ............................................................................ 15
2.2.12.3 Silver Staining..................................................................................................... 16
2.2.13 Statistical Analysis......................................................................................................... 16


3 Results.............................................................................................................................................. 17

3.1 Antibody Array Technologies.................................................................................................. 17
3.1.1Cy3 and Cy5 of SDS-PAGE, Microscope-Slide-Size Array, Mini-Arrayslide...................... 17
3.1.2 Data Analysis .................................................................................................................... 20

3.2 Modulation of Phosphorylation and Localization in BMP2 Treated U937
Cells................................................................................................................................................. 26
3.2.1 Total p-Tyrosine and p-p38 Increased: Cytoplasm and Nucleus Increasing....................... 29
3.2.2 Total p-ERK and p-JNK Increased: Cytoplasm Increasing and Nucleus Unchanged........ 32
3.2.3 Total p-Akt Increased: Cytoplasm Increasing and Nucleus Decreasing.... ........................ 34
3.2.4 Total p-Smad1, p-Smad2/3 and c- Myc Unchanged: Cytoplasm Decreasing
and Nucleus Increasing...................................................................................................... 35
3.2.5 Total p-p70S6 Unchanged: Cytoplasm Increasing and Nucleus Decreasing..................... 39



3.3 Modulation of Phosphorylation and Localization In BMP2 Treated MCF7 Cells .............. 40

3.3.1 Total p-JNK, p-Smad1, p-Smad2/3, p-p38 and p38 Unchanged: Cytoplasm Increasing and
Nucleus Decreasing...........................................................................................................

3.3.2 p-Tyrosine, p-STAT3, p-ERK and p-P70S6 Increased: Cytoplasm Increasing and nucleus
unchanged ...................................................................................................................................
43
3.3.3Total c-Myc Unchanged: Nucleus Slightly Increasing and Cytoplasm Slightly
Decreasing.....................................................................................................................…………. 46

3.3.4 Total p-Akt Unchanged: Nucleus and Cytoplasm unchanged............………………………….47

3.4 Modulation of Phosphorylation and Localization In STI Treated K562 Cells ................... 47

3.4.1 Total p-Akt and p-p70S6 Decreased: Cytoplasm and Nucleus Decreasing....................... 47
3.4.2 Total p-p38, p38, c-Myc, p-Tyrosine, p-Smad1 and p-Smad2/3 Increased:
Cytoplasm and Nucleus Increasing.................................................................................. 48
3.4.3Total p-STAT3 and p-JNK Increased: Cytoplasm Increasing and Nucleus
Unchanged............................................................................................................................................. 48
3.4.4 Total p-ERK Increased: Cytoplasm Decreasing and Nucleus Increasing.. ......................... 48

4 Discussion........................................................................................................................................ 52

4.1 Antibody Array Technologies.................................................................................................. 52

4.2 BMP2 Signalling In U937 Cells................................................................................................. 54
4.2.1 BMP2 activates Akt, ERK and JNK pathway by increase of cytoplasmic phosphorylation in
U937 cells............................................................................................................................................... 54
4.2.2 BMP2 activates Smad1 and Smad2/3 network, p38 network and c-Myc, Tyrosine network by
increase of nuclear phosphorylation in U937 cells...................................................................................55
4.2.3 BMP2 inhibits p70S6 signalling by increase of cytoplasmic phosphorylation and decrease of
nucleus in U937 cells...............................................................................................................................57
4.2.4Possible intracellular relationship in BMP2 treated U937 cells........................................