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| | RSK2 Inhibition Assay |
| Description: | The inhibition assays against RSK2 in vitro were determined by ADP Quest® (DiscoveRx) in 96-well flat-bottom plates according to the manufacturer's instructions [Lu et al., J. Med. Chem., 54:3564-3574]. The kinase (20 ng millipore) in 40 μL assay buffer (15 mM HEPES, pH 7.4, 20 mM NaCl, 1 mM EGTA, 0.02% Tween 20, 10 mM MgCl2, and 0.1 mg/mL BGG) containing 25 μM S6 peptide (AKRRRLSSLRA, Anaspec) was incubated for 20 min at room temperature with indicated concentrations of the compounds to be tested. Reactions were initiated by adding 10 μL of ATP to a final ATP concentration of 10 μM and terminated after 60 min at room temperature by adding 20 μL ADP Reagent A and 40 μL ADP Reagent B. Ro31-8220 was used as a positive control. Compounds dilutions were prepared from stock in DMSO and diluted with assay buffer for inhibition assay. The fluorescence signal detecting the amount of ADP produced as a result of enzyme activity was recorded on the Synergy™ 2 Multi-Mode Microplate Reader (BioTek) at an excitation wavelength of 530 nm and an emission wavelength of 590 nm at 30 min after the addition of the ADP Reagent B. |
| Affinity data for this assay | |
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