Chapter 1
Quantitative Immunofluorescence for Measuring Spatial Compartmentation of Covalently-Modified Signaling Proteins

1.1.		Introduction
1.2.		Experimental Design
1.3.		Materials
1.3.1.		Cell Culture
1.3.2.		Buffers/Reagents
1.3.3.		Immunofluorescence Reagents
1.4.		Methods
1.4.1		Cell culture and stimulation for phospho-ERK measurements
1.4.2		Antibody labeling of ppERK
1.4.3		Fluorescence Microscopy Imaging of ppERK and Automated Image analysis
1.5.		Data Acquisition, Anticipated Results, and Interpretation
1.6.		Statistical Guidelines
1.7.		Discussion and Commentary
1.8.		Troubleshooting Table
1.9.		Application Notes
1.10.		Summary points
1.11.		Acknowledgements
1.12.		References

Chapter 2
Development of Green Fluorescent Protein-Based Reporter Cell Lines for Dynamic Profiling of Transcription Factor and Kinase Activation

2.1.		Introduction
2.2.		Materials
2.2.1.		Cell and Bacterial Structure
2.2.2		Buffers and Reagents
2.2.3.		Cloning
2.2.4		Microscopy
2.3.		Methods
2.3.1		3T3-L1 Cell Culture
2.3.2		Transcription Factor Reporter Development
2.3.2.1		Identification of Response Elements
2.3.2.2		Identification of TF Binding Sites
2.3.2.3		Cloning TF Binding Elements into Reporter Plasmid
2.3.3.		Kinase Reporter Development
2.3.3.2		Selection of FRET Elements
2.3.3.2		Fluorescent Protein PCR
2.3.3.3		Fluorescent Protein Cloning
2.3.3.4		Linker Oligonucleotide Development and Annealing
2.3.3.5		Linker Region Cloning
2.3.3.6		FRET Control Plasmid Development
2.5.		Application Notes
2.5.1		Electroporation of TF Reporter Plasmids into 3T3-L1 Preadipocytes
2.5.1.1		Electroporation of TF Reporter Plasmids into 3T3-L1 Preadipocytes
2.5.1.2		Clonal Selection
2.5.1.3		Clonal Screening
2.5.1.4		Monitoring PPARγ Activation in 3T3-L1 Adipocytes
2.5.2		Monitoring Activation of ERK in HepG2 Cells
2.6.		Data Acquisition, Anticipated Results, and Interpretation
2.7.		Discussion and Commentary
2.8.		Troubleshooting Table
2.9.		Summary Points
		Acknowledgements
		References

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