.... Fluorescein at 0.01 mM, pH 9 was used as a lifetime reference standard. The FRET efficiency was calculated as previously described ( Khan et al., 2017 ). HeLa cells were seeded onto a six-well plate. The following day, these cells were transfected with Renilla Luciferase control vector (pRLTK), NF-κB...

... of the living organism. Advanced FRET probes allow tracking signaling events in live cells. Light-sheet microscopy allows for deeper tissue penetration in optically clear samples, enriching our understanding of the higher-level organization of the immune response. Super-resolution microscopy offers insights...

...-FRET and FLIM-FRET. PIP 3 -mediated association with the cytoplasmic membrane and non-phosphorylated Y426 are required for SWAP-70 to dimerize and to bind F-actin. The dimerization region was identified near the C terminus where R546 is required for dimerization and, thus, F-actin bundling...

... neurons from McGill-R-Thy1-APP transgenic rats, a model of Alzheimer's disease-like amyloid pathology. Live FRET imaging of neurons from transgenic rats revealed perturbed ‘lipid-MERCs’ (gap width <10 nm), while ‘Ca 2+ -MERCs’ (10–20 nm gap width) were unchanged. In situ TEM showed no significant...

...Sonal Prasad; Evgeni Ponimaskin; Andre Zeug ABSTRACT Protein–protein interaction is often investigated using quantitative molecular microscopy with Förster resonant energy transfer (FRET). Here, we combined ‘linear unmixing FRET’ (lux-FRET) with the simultaneous application of a FRET-based...

... flow cytometry (FACS canto, BD Biosciences) and sorting of matched-expressing cell lines (using FACSAria II or MoFlo) were conducted within the Biomedical Research Facility at King's College London. where ηFRET is the FRET efficiency, R0 is the Förster radius, r is the molecular separation...

... in a yeast-two hybrid screen of a cardiac library. We corroborated this interaction by performing Förster resonance energy transfer (FRET) in HEK293 cells and co-immunoprecipitation in HEK293 cells and native cardiac tissue. TRIOBP-1 overexpression reduced hERG surface expression and current density, whereas...

... these hurdles. As discussed above, intravital FRAP and FRET imaging can provide pivotal insights into the subcellular regulation of protein dynamics and activity. However, these approaches are limited by the optical restrictions of confocal laser scanning and multiphoton microscopy, including magnification...

... of adequate amplitude to activate chloride secretion. Cystic fibrosis Correctors cAMP PKA Airway cells FRET The most prevalent mutation of the CFTR gene associated with cystic fibrosis is the deletion of phenylalanine at residue 508 (F508del CFTR) in the nucleotide-binding domain 1 (NBD1...

... a newly developed FRET sensor. A combination of a Rab7 sensor and confocal FRET imaging techniques revealed that the activation of Rab7 on late endosomes depends on Mon1–Ccz1 and is implicated in late-endosome–lysosome fusion. In contrast, Rab7 activity on lysosomes was independent of Mon1–Ccz1 and active...

... regulates the KCNQ2 channel through a dual rearrangement of cytosolic termini that underlies upregulation of single-channel gating and a reduction in the number of functional channels. FRET KCNQ2 Src Gating Open probability M-channels are members of the Kv7 family of voltage-dependent K...

.... Nevertheless, the dynamics of phosphatidylinositol-(3,4,5)-trisphosphate (PIP 3 ) and the regulation of its synthesis and degradation at synaptic compartments is far from clear. Here, we have used fluorescence resonance energy transfer (FRET) imaging to monitor changes in PIP 3 levels in dendritic spines from...

... they bind effectors. Previous studies have suggested that the binding of Rac1 to membranes requires, and colocalizes with, cholesterol-rich liquid-ordered ( lo ) membrane domains (lipid rafts). Here, we have developed a fluorescence resonance energy transfer (FRET) assay that robustly detects Rac1 membrane...

...). Using high-resolution microscopic imaging, we find that cyclin A2 persists beyond metaphase. Indeed, we identify a novel cyclin-A2-containing compartment that forms dynamic foci. Förster (or fluorescence) resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM) analyses show...

... and bind to specific DNA sequences, resulting in target gene expression. We analysed and compared the structural organizations of the unphosphorylated latent and phosphorylated activated STAT3 dimers using Förster resonance energy transfer (FRET) in fixed and living cells. The latent dimers are stabilized...

... hair cells than in outer hair cells, and began as stereocilia fusion and depletion, accompanied by a thinning and waving circumferential actin belt at apical junctional complexes (AJCs). Adenovirus-encoded GFP–Cdc42 expression in hair cells and fluorescence resonance energy transfer (FRET) imaging...

... ( Day and Davidson, 2009 ; Sample et al., 2009 ; Shaner et al., 2005 ) enabled the construction of FRET-based biosensors, with changes in FRET actively reporting on biochemical events in cells. The first such biosensor, the Ca 2+ probe Cameleon, featured CaM and the M13 peptide sandwiched between...

... of these CaM-binding regions, we generated a fluorescence resonance energy transfer (FRET) pair, a dual CFP- and YFP-labeled RyR2 (RyR2 R3595-CFP/K4269-YFP ) with CFP inserted after Arg3595 and YFP inserted after Lys4269. We transfected HEK293 cells with the RyR2 R3595-CFP/K4269-YFP cDNA, and examined...

... © 2013. Published by The Company of Biologists Ltd 2013 Islet β-cells Apoptosis Necrosis Live-cell imaging Fluorescence resonance energy transfer FRET For cell death assays, MIN6 and dispersed islet cells were seeded into 96-well plates and infected with lentiviral particles...

... changes in Syx by FRET. We explored the recent crystal structure of Syx in order to rationalize our design of the probes. In the available structure, the N-terminus of Syx and its juxtamembrane region, connecting the transmembrane anchor and the H3 domain, are in proximity in the ‘closed’ rather than...