| Assay Method Information | |
| | MK2 Enzyme Inhibition Assay |
| Description: | For MK2 inhibition, reactions contained compound or vehicle control, hsp27 peptide substrate and activated MK2 mix containing ATP. After incubation, samples were transferred to black 384-well plates for detection of phosphorylated hsp27 by time-resolved fluorescence resonance energy transfer using an antibody mix containing a rabbit anti-phospho-hsp27 (Ser82) antibody, and anti-rabbit europium-labeled secondary antibody LANCE Eu-W1024 (Perkin Elmer) as fluorescence donor along with streptavidin SureLight-APC (Perkin Elmer) as acceptor. Following incubation at room temperature for 90 min, the FRET ratio 665/620 nm was determined. Individual IC50 values of compounds were determined by nonlinear regression after fitting of curves to the experimental data using Excel XL fit 4.0 (Microsoft). |
| Affinity data for this assay | |
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