![]() Coelenterazine was added to a final concentration of 2 μ m immediately before measuring BRET. Inhibitors were added 5 min before the indicated treatments. 100 μl was added to 96-well white plates and treated for 5 min at room temperature with the indicated treatments. After centrifugation at 20,000 × g for 10 min, the supernatant was removed and diluted to the desired volume. Guanine Nucleotide Exchange Factor (GEF).Bioluminescence Resonance Energy Transfer (BRET).The results reveal a rapid means to identify modulators (potentially including allosteric inhibitors) of Epac activity that also provides insight into the mechanisms of Epac activation and inhibition. We used computational molecular modeling to analyze the interaction of analogs with Epac1. ![]() To confirm the results with the CAMYEL assay, we used Swiss 3T3 cells and assessed the ability of cyclic nucleotide analogs to modulate the activity of Epac or PKA, determined by Rap1 activity or VASP phosphorylation, respectively. The CAMYEL assay can also identify competitive and uncompetitive Epac inhibitors, e.g. We found that the use of CAMYEL can detect the binding of cAMP analogs to Epac and their modulation of its activity and can distinguish between agonists (cAMP), partial agonists (8-chlorophenylthio-cAMP), and super agonists (8-chlorophenylthio-2′- O-Me-cAMP). Using a bioluminescence resonance energy transfer-based assay (CAMYEL) to examine modulators of Epac activity, we took advantage of its intramolecular movement that occurs upon cAMP binding to assess Epac activation. Epac, a guanine nucleotide exchange factor for the low molecular weight G protein Rap, is directly activated by cAMP. Epac has been implicated in many responses in cells, but its precise roles have been difficult to define in the absence of Epac inhibitors. The signaling molecule cAMP primarily mediates its effects by activating PKA and/or exchange protein activated by cAMP (Epac). Glycobiology and Extracellular Matrices.
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