| Assay Method Information | |
| | Electrophoretic Mobility Shift Assay (EMSA) |
| Description: | DNA binding reaction (20 μL) was carried out in 1×DNA Binding Buffer (10 mM Tris, 40 mM NaCl, 1 mM EDTA, 4% glycerol) with 10 nM c-Rel protein and 0.5 ng phosphor-labeled CD28RE oligonucleotide for 10 minutes at room temperature in 96-well plates. Test compounds, in serial dilutions, were added into each well, and further incubated for 15 minutes before loading onto native 5% polyacrylamide gel. Electrophoresis proceeded for 2.5 hours at 160V. Radioactive signals were quantified using Phospho-Imager. IC50 of compounds of example 1 to 75 were determined by quantifying the intensity of Rel/NF-κB inhibition in EMSA using phospho-imager. |
| Affinity data for this assay | |
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