Journal: iScience

Article Title: Robust calibration and quantification of FRET signals using multiplexed biosensor barcoding

doi: 10.1016/j.isci.2025.113743

Figure Lengend Snippet: Construction of FRET calibration standards (A) Construction of FRET-ON calibration standard by Y349F mutation of Cyto-FAK. (B) Plot of YFP/CFP ratio of CytoFAK and FRET-ON (mean ± SEM, n = 20 cells) in response to 100 ng/mL EGF added at 6 min. (C) Plot of YFP/CFP ratio of CytoFAK and FRET-ON (mean ± SEM, n = 20 cells) in response to FAK inhibitor VS-6063 over 24 h. (D) Construction of FRET-OFF calibration standard by removal of the H3 domain of the H3K9me9 (W45A) biosensor. (E) Plot of YFP/CFP ratio of FRET-OFF and H3K9me3 biosensor (mean ± SEM, n = 20 cells) in response to 5 μM TCP over 48 h.

Article Snippet: H3K9me3 (W45A) biosensor , Addgene , Cat#120808.

Techniques: Mutagenesis

Journal: Science Advances

Article Title: Mechanochemical waves in focal adhesions during cell migration

doi: 10.1126/sciadv.adw6425

Figure Lengend Snippet: ( A ) The FAK biosensor is composed of ECFP, SH2 domain, flexible linker, FAK substrate peptide, YPet, and FAT domain. ( B ) Schematics illustrating the FRET effect of the FAK biosensor upon the actions of FAK phosphorylation or dephosphorylation. Upon phosphorylation of Y397 in the biosensor FAK substrate peptide, the SH2 domain forms an intramolecular complex with the phosphotyrosine side chain, increasing the distance between the FRET pair to alter the FRET signal. Dephosphorylation reverses the FRET change. ATP, adenosine 5′-triphosphate. ( C ) C-terminal FAT domain recruits the biosensor to FAs. YPet intensity showing slight changes before and after FAK inhibition (FAKi; 10 μM PF-573228, 60 min). ( D ) ECFP/FRET signal before and after FAKi (10 μM PF-573228, 60 min) showing that the biosensor is specific and sensitive to FAK activity. ( E ) EFCP/FRET signal at individual FAs [ n = 195 FAs from seven cells across three independent experiments for FAKi (10 μM PF-573228, >60 min); n = 151 FAs from six cells across three independent experiments for DMSO control; means ± SD]. ( F ) Fluorescence lifetime image and quantification for fibroblasts expressing the FAK biosensor ( n = 23 FAs from six cells across three independent experiments). Scale bar, 20 μm. ( G ) FRET efficiency image and quantification for FAs and cytosol ( n = 19 FAs from five cells across three independent experiments). Scale bar, 20 μm. a.u., arbitrary units.

Article Snippet: The FAK FRET biosensor construct is available from Addgene (plasmid no. 78303).

Techniques: Phospho-proteomics, De-Phosphorylation Assay, Inhibition, Activity Assay, Control, Fluorescence, Expressing