| Assay Method Information | |
| | The p38alpha:MK2:HSP27 biochemical assay |
| Description: | In a 20 μL reaction, the compounds of various concentrations (20 nL) were pre-incubated with 0.6 nM MK2 (Abcam #ab79910) and 0.06 nM active p38α (Abcam #ab271606 or in-house prepared: Avi-Tev-8HIS-p38a phosphorylated by GST-MKK6) for 15 minutes at room temperature following by the addition of 2 μMH SP27 peptide substrate ((TAMRA)Cys-KKKALSRQLSVAA) (custom synthesized, Elim Biopharmaceuticals) and 10p M ATP (Fisher Scientific #B20). After 1 h incubation at room temperature, 60 μL detection reagent (Molecular Devices #8160) was added to the reaction and incubate d for 12 h at room temperature. Homogeneous Time Resolved Fluorescence (HTRF) signal (the ratio between emission at 550 nm and emission at 570 nm upon excitation at 330 nm) was read by TECAN Spark microplate reader. The signal was normalized to vehicle control (DMSO, 0% inhibition) and 1 μM GS-703447 (100% inhibition) to generate % inhibition as a function of compound concentration and fitted in a four-parameter logistic equation to generate IC50. |
| Affinity data for this assay | |
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