ABSTRACT
Introduction............................................................................................................240 The 14-3-3 Protein Acts as a Molecular Adaptor in Signaling Networks ...............................................................................240 The Advantages of Protein Microarray Analysis to Identify Protein-Protein Interactions ....................................................240
Experimental Protocols..........................................................................................241 Preparation of an Epitope-Tagged Probe for Microarray Analysis ..........241 Protein Microarray Analysis......................................................................242
Validation and Evaluation of the Results of Protein Microarray Analysis ..........245 Transient Expression of Recombinant Proteins in HEK293 Cells...........245 Coimmunoprecipitation Analysis ..............................................................246 Bioinformatic Analysis ..............................................................................246
Results....................................................................................................................247 Protein Microarray Analysis Identified 20 Distinct 14-3-3-Interactors ...................................................................247 Immunoprecipitation Analysis Validated the Specific Binding to 14-3-3 ..................................................................250
Discussion ..............................................................................................................251 Protein Microarray Analysis Effectively Identifies 14-3-3-Binding Proteins ............................................................................251 Potential Problems Remain to Be Solved in the Present Study...................................................................................251 Biological Roles of 14-3-3-Interacting Proteins .......................................252
Future Directions ...................................................................................................253
Summary and Conclusions....................................................................................254 Acknowledgments..................................................................................................254 References..............................................................................................................254
The 14-3-3 protein family in mammalian cells consists of evolutionarily conserved, acidic 30-kDa proteins composed of seven isoforms named
