| Solution Information | help | |
| Enzyme: | Eukaryotic translation initiation factor 2-alpha kinase 3 | |
| inhibitor: | BDBM46491 | |
| substrate: | n/a | |
| Solution Type: | Aqueous | |
| pH at Preparation: | n/a | |
| Temp. Prep.: | n/a | |
| Comments: | Assay Overview: The purpose of this assay is to determine dose response curves for compounds identified as active in a previous set of experiments entitled, "Primary cell-based high-throughput screening assay to measure PERK inhibition" (PubChem AID 1416). The assay employs a CHO cell line that stably expresses PERK's cytoplasmic kinase domain fused to a modified dimerization domain (DD). Treatment of the cells with the ligand AP20187 induces dimerization of the DD domains, leading to PERK kinase activation, eIF2a phosphorylation, and repression of protein translation. Stable co-expression of a plasmid encoding secreted alkaline phosphatase (SEAP) enables protein translation to be monitored using a SEAP chemiluminescence substrate, which produces light when dephosphorylated by SEAP. As designed, test compounds that inhibit dimerizer-induced PERK activity will prevent PERK-mediated eIF2a phosphorylation and repression of translation, leading to increased SEAP levels, increased SEA | |
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