In this study, three-channel microscopy was used to study the effect of lipid rafts disruption on LMP1 oligomerization by quantitative fluorescence resonance transfer method (E-FRET). We first verified four crosstalk and bleedthrough parameters and the system parameter G on our imaging system by ECFP-only and EYFP-only plasmids. Furtherly, two FRET-based probes associated with LMP1, were constructed to study oligomerization of oncoprotein LMP1 in nasopharyngeal carcinoma cell line (CNE1). Methyl-β-cyclodextrin (MβCD) was used to disrupt lipid rafts, quickly. The FRET images displayed that majority of LMP1 oligomer localized in internal perinuclear membranes of CNE1 and enhancement of LMP1 oligomerization caused by lipid rafts disruption. These findings provided some novel views for LMP1 and lipid rafts.
Adenosine plays important roles in the pain signal transduction by activating adenosine receptors of two subtypes of A1 and A2A. In this study, FRET system based independent emission -spectral spectral spectral unmixing method (Iem-spFRET) was set up and used to measure the energy transfer from A1R to A2AR. The energy transfer efficiency calculated by Iem-spFRET is about 17.44%. All the above date and results demonstrate that FRET with special designed fluorescence proteins could be used to investigate the interaction between adenosine receptors.
In this study, two FRET-based probes are constructed to research oligomerization of Epstein-Barr virus Oncoprotein LMP1 in live cells. The images of wide-field fluorescence microscopy display that the majority of two LMP1-associated probes co-localized in internal perinuclear membranes. Furthermore, the fluorescence spectra of single cell co-expressed two probes indicated that the ratio of two emission peaks is around one, and the fluorescence spectra changed insignificantly during an hour observation. These findings indicated that LMP1/LMP1 interacted stably in live cells.
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