Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay
Abstract
:1. Introduction
2. Results
2.1. Establishment of FRET Measurements by Targeting of Non-Interacting, Cytosolic Proteins to Membranes
2.2. Homotypic and Heterotypic Interactions between IFITM Proteins
2.3. F75 and F78 Are Not Required for Normal Membrane Targeting of IFITM3 and for IFITM3/IFITM3 Interactions
2.4. F75 and F78 are Dispensable for IFITM3-IFITM3 Interactions as Determined by Co-Immunoprecipitation
3. Discussion
4. Materials and Methods
4.1. Cell Culture
4.2. Plasmid Construction
4.3. Oligonucleotides
4.4. Antibodies
4.5. Immunofluorescence
4.6. Immunoblot
4.7. Production of Retroviral Vectors and Transduction Experiments
4.8. Coimmunoprecipitation
4.9. Flow-Cytometry and FRET
Supplementary Materials
Author Contributions
Funding
Acknowledgments
Conflicts of Interest
Abbreviations
EBOV | Ebola virus |
eCFP | enhanced cyan fluorescent protein |
eYFP | enhanced yellow fluorescent protein |
FLUAV | Influenza A virus |
FRET | Fluorescence resonance energy transfer |
IFITM | Interferon-induced transmembrane protein |
MLV | Murine leukemia virus |
SCFP | super cyan fluorescent protein |
SYFP | super yellow fluorescent protein |
wt | wildtype |
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Winkler, M.; Wrensch, F.; Bosch, P.; Knoth, M.; Schindler, M.; Gärtner, S.; Pöhlmann, S. Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay. Int. J. Mol. Sci. 2019, 20, 3859. https://doi.org/10.3390/ijms20163859
Winkler M, Wrensch F, Bosch P, Knoth M, Schindler M, Gärtner S, Pöhlmann S. Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay. International Journal of Molecular Sciences. 2019; 20(16):3859. https://doi.org/10.3390/ijms20163859
Chicago/Turabian StyleWinkler, Michael, Florian Wrensch, Pascale Bosch, Maike Knoth, Michael Schindler, Sabine Gärtner, and Stefan Pöhlmann. 2019. "Analysis of IFITM-IFITM Interactions by a Flow Cytometry-Based FRET Assay" International Journal of Molecular Sciences 20, no. 16: 3859. https://doi.org/10.3390/ijms20163859