| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | For the construction of the Lyn-GFP10, the coding sequence of the first 11 residues (MGCIKSKGKDS) of the human Lyn-kinase and the full coding region of GFP1056 (Supplementary Data 1) were synthesized at GenScript (Piscataway, NJ) and subcloned into pcDNA 3.1/zeo (−) using infusion (Clontech, CA). A humanized rGFP (Supplementary Data 1) was synthetized at GenScript (Piscataway, NJ). The Lyn-rGFP was generated by replacing the coding sequence of GFP10 in the Lyn-GFP10 construct by rGFP, which was generated by PCR amplification. StreptagII-fused GFP10 was synthesized at GenScript and subcloned into pcDNA 3.1/zeo (−) (STII-GFP10). | Get A Quote |
Endocytosis and intracellular trafficking of receptors are pivotal to maintain physiological functions and drug action; however， robust quantitative approaches are lacking to study such processes in live cells. Here we present new bioluminescence resonance energy transfer (BRET) sensors to quantitatively monitor G protein-coupled receptors (GPCRs) and β-arrestin trafficking. These sensors are based on bystander BRET and use the naturally interacting chromophores luciferase (RLuc) and green fluorescent protein (rGFP) from Renilla. The versatility and robustness of this approach are exemplified by anchoring rGFP at the plasma membrane or in endosomes to generate high dynamic spectrometric BRET signals on ligan... More
