Front Cover; Molecular Characterization of Autophagic Responses, Part B; Copyright; Contents; Contributors; Preface; 1. Introduction; Acknowledgments; References; Chapter One: Renilla Luciferase-LC3 Based Reporter Assay for Measuring Autophagic Flux; 1. Introduction; 1.1. The Challenge of Measuring Autophagic Flux; 1.2. LC3 as a Marker of Autophagy; 1.3. Cautions in Using LC3-II Turnover as a Marker of Autophagic Flux; 2. The Rluc-LC3 Assay; 2.1. Description of the Assay; 2.2. Applications of the Rluc-LC3 Assay; 3. Designing the Rluc-LC3wt and Rluc-LC3G120A Fusion Proteins

4. Establishing Rluc-LC3wt- and Rluc-LC3G120A-Expressing Cells4.1. Expression Vectors and Cell Lines; 4.1.1. Stable Transfection of MCF7 Cells (Transfections Are Performed Mainly According to the Fugene HD Protocol (Promega ... ; 5. The Rluc-LC3 Assay Performed on Cell Lysates; 5.1. Buffers and Solutions; 5.2. Protocol; 6. The Rluc-LC3 Assay Performed on Live Cells; 6.1. Buffers and Solutions; 6.2. Protocol; 6.3. Notes; Acknowledgments; References; Chapter Two: Measurement of Autolysosomal pH by Dual-Wavelength Ratio Imaging; 1. Introduction; 2. Cell Preparation

3. Equipment Setup and Software Requirements4. Image Acquisition; 5. In Situ pH Calibration; 6. Image Analysis and Determination of Autolysosomal pH; 7. Conclusion; Acknowledgments; References; Chapter Three: Long-Lived Protein Degradation During Autophagy; 1. Introduction; 2. Proteolysis; 2.1. Materials and Reagents; 2.2. Protocol (see Fig. 1); 2.3. Notes and Potential Pitfalls; 3. Other Methods for Measuring Autophagic Flux; 4. Conclusion; Acknowledgments; References

Chapter Four: Proteomic Profiling of De Novo Protein Synthesis in Starvation-Induced Autophagy Using Bioorthogonal Noncan ... 1. Introduction; 2. Materials, Equipment, and Solutions; 2.1. Materials; 2.2. Equipment; 2.3. Methionine-Free DMEM; 2.4. Amino Acid-Free Medium; 2.5. C18 Buffers; 2.6. Gradient Separation Buffer; 3. Fundamentals: AHA Labeling Combined With the iTRAQ Approach for Identification of De Novo Protein Synthesis; 3.1. General Principles; 3.2. Advantages of This Approach; 3.3. Applications of This Approach; 4. Protocol; 4.1. Cell Culture and Metabolic Labeling by AHA

4.2. Click Chemistry Tagging With Biotin Alkyne4.3. Avidin Affinity Purification; 4.4. Isobaric Tag for Relative and Absolute Quantification (iTRAQ) Labeling; 4.5. Sample Cleanup by Strong Cation Exchange Chromatography; 4.6. Desalting of Labeled Samples by the C18 Column; 4.7. Nano-LC Electrospray Ionization MS; 4.8. Protein Identification and Quantification Using ProteinPilotTM Software; 5. Limitations; References; Chapter Five: Methods to Monitor and Manipulate TFEB Activity During Autophagy; 1. Introduction; 2. Methods to Monitor TFEB/TFE3 Activation