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Chapter 1 Introduction 1

1.1 Antibody Array Technologies and Applications 1

1.2 Protein Phosphorylation,Localization and Function 3

1.3 Interaction Proteomics and Pathway Building 4

1.3.1 BMP2 Signaling 5

1.3.2 The Effect of STI571 on Cell Signaling 11

Chapter 2 Technologies 12

2.1 Materials 12

2.1.1 Cell Lines 12

2.1.2 Chemicals 12

2.1.3 Antibodies 12

2.1.4 Protein-Molecular Weight Marker 13

2.1.5 Apparatus 13

2.2 Methods 14

2.2.1 Cell Culture 14

2.2.2 Cell Numbers and Experimental Format 14

2.2.3 Cytoplasmic and Nuclear Protein Extracts 14

2.2.4 Determination of Protein Concentration 15

2.2.5 Ponceau S Staining 15

2.2.6 Western Analysis 16

2.2.7 Immunoprecipitation 16

2.2.8 Cell Cycle Analysis 16

2.3 Silver Staining in ArrayTube 17

2.3.1 Biotinylation of Cellular Proteins 17

2.3.2 Incubation of Protein Extracts 17

2.3.3 Silver Staining 17

2.3.4 Statistical Analysis 18

Chapter 3 New Cytoplasmic and Nuclear BMP2-Signaling Pattern in U937 Cells 23

3.1 Modulation of Phosphorylation and Localization in BMP2-Treated U937 Cells 23

3.1.1 Total p-Tyrosine and p-p38,p38 Increased:Increasing Cytoplasm and Nucleus 23

3.1.2 Total p-ERK and p-JNK Increased:Increasing Cytoplasm and Unchanged Nucleus 25

3.1.3 Total p-Akt Increased:Increasing Cytoplasm and Decreasing Nucleus 26

3.1.4 Total p-Smad1,p-Smad2/3,c-Myc and p-STAT3 Unchanged:Decreasing Cytoplasm and Increasing Nucleus 29

3.1.5 Total p-p70S6 Unchanged:Increasing Cytoplasm and Decreasing Nucleus 32

3.2 BMP2 Signaling in U937 Cells 32

3.2.1 BMP2 Activates Akt,ERK and JNK Pathway in U937 Cells 33

3.2.2 BMP2 Activates Smad1 and Smad2/3 Networks,p38 Network and c-Myc,Tyrosine Network in U937 Cells 35

3.2.3 BMP2 Inhibits p70S6 Signaling in U937 cells 36

3.3 BMP2 Signaling Model in U937 Cells 38

Chapter 4 New Cytoplasmic and Nuclear BMP2-Signaling in MCF7 Cells 40

4.1 Modulation of Phosphorylation and Localization in BMP2-Treated MCF7 Cells 40

4.1.1 Total p-JNK,p-Smad1,p-Smad2/3,p-p38 and p38 Unchanged:Increasing Cytoplasm and Decreasing Nucleus 41

4.1.2 p-Tyrosine,p-STAT3,p-ERK and p-p70S6 Increased:Increasing Cytoplasm and Unchanged Nucleus 45

4.1.3 Total c-Myc Unchanged:Slightly Increasing Nucleus and Slightly Decreasing Cytoplasm 46

4.1.4 Total p-Akt Unchanged:Unchanged Nucleus and Cytoplasm 46

4.2 BMP2 Signaling in MCF7 Cells 48

4.2.1 BMP2 Activates p70S6 and ERK Signaling,Tyrosine and STAT3 Network in MCF7 Cells 48

4.2.2 BMP2 Inhibits p38,JNK and Smad Signaling in MCF7 Cells 49

4.2.3 BMP2 Activates c-Myc Signaling in MCF7 Cells 50

4.3 BMP2 Signaling Model in MCF7 Cells 50

Chapter 5 New Cytoplasmic and Nuclear STI571 Signaling in K562 Cells 52

5.1 Modulation of Phosphorylation and Localization in STI571-Treated K562 Cells 52

5.1.1 Total p-Akt and p-p70S6 Decreased:Decreasing Cytoplasm and Nucleus 52

5.1.2 Total p-p38,p38,c-Myc,p-Tyrosine,p-Smad1 and p-Smad2/3 Increased:Increasing Cytoplasm and Nucleus 55

5.1.3 Total p-STAT3 and p-JNK Increased:Increasing Cytoplasm and Unchanged Nucleus 56

5.1.4 Total p-ERK Increased:Decreasing Cytoplasm and Increasing Nucleus 58

5.2 STI571 Signaling in K562 Cells 58

5.2.1 STI571 Inhibits Akt/p70S6 PI3K Signaling in K562 Cells 58

5.2.2 STI571 Activates p38 MAPK Pathway,c-Myc,Tyrosine and Smad1/Smad2/Smad3 Networks in K562 Cells 59

5.2.3 STI571 Activates ERK Pathway in K562 Cells 59

5.2.4 STI571 Activates STAT3 Network and JNK/MAPK Pathway in K562 Cells 60

5.3 STI571 Signaling Model in K562 Cells 61

Chapter 6 Positive and Negative Co-localized Relationships in Three Treated Cell Lines 63

6.1 Positive and Negative Co-localized Distributions 63

6.1.1 p-p38 and p38 63

6.1.2 Smad1 and Smad2/3 63

6.1.3 Akt,ERK and p70S6 63

6.1.4 p70S6 with p38 and Smad1,2,3 75

6.1.5 ERK or p38 with Smad1 80

6.1.6 ERK or p38 with c-Myc 81

6.2 Possible Relationships 83

Chapter 7 Cytoplasmic Signaling in Three Treated Cell Lines 87

7.1 Cytoplasmic Distribution 87

7.1.1 p-ERK,p-JNK and p-Akt in BMP2-Induced Apoptosis in U937 Cells 87

7.1.2 p-STAT3 and p-JNK in STI571-Treated K562 Cells 87

7.1.3 p-Tyrosine,p-STAT3,p-ERK and p-p70S6 in BMP2-Treated MCF7 Cells 91

7.2 Co-cytoplasmic Signaling 92

Chapter 8 Proteomic Silencing in Treated Human Cancer Cell Lines 95

8.1 Proteomic Silence Distributions 95

8.1.1 p-p70S6 Nuclear Decrease in BMP2-Induced Apoptosis in U937 Cells 95

8.1.2 p-ERK/p-JNK/p-p38,p-Smad1 and p-Smad2/3 Co-nuclear Decrease in BMP2-Treated MCF7 Cells 98

8.1.3 p-Akt and p-p70S6 Co-cytoplasmic and Co-nuclear Decrease in STI571-Treated K562 Cells 98

8.2 Possible Relationships 98

Chapter 9 Signaling Pathway Setting-up Based on Bioinformatics 100

9.1 Methods 100

9.1.1 Mean and Variance 100

9.1.2 Hierarchical Clustering 101

9.1.3 Competitive Learning Network 101

9.1.4 Self-Organizing Map(SOM) 104

9.2 Results 106

9.2.1 Mean and Variance Analysis of Cytoplasmic Signaling in Control and STI571-Treated K562 cells 106

9.2.2 Competitive Learning Network Analysis of Cytoplasmic Signaling in Control and STI571-Treated K562 cells 107

9.2.3 Hierarchical Clustering Analysis of Cytoplasmic Signaling in Control and STI571-Treated K562 cells 109

9.2.4 SOM Analysis of Cytoplasmic Signaling in Control and STI571-Treated K562 cells 110

9.3 Discussion 117

9.4 Conclusions 118

References 119