External ID: 20160513eIF4E

Protocol: The cells carrying the bicitronic reporter gene (E8-Luc) were plated in 96-well plates at a density of 1.2x10(5)/well (100 ul DMEM) overnight. 0.5 ul of tested compounds (0.5 mM) or DMSO were added into the plates, and treated the cells for 16 h. The plates were then washed with 100 ul of PBS twice using Aquamax Plate Washer (Molecular Devices), and lysated in 60 ul of lysis buffer (1% Triton X-100, 25 mM Gly-Gly, pH 7.8, 15 mM MgSO4, and 4 mM EGTA) at room temperature for 20 min. 10 ul of lysates were then subjected to luciferase activity assays using Spectramax L (Molecular Devices) and the Promega Luciferase Substrate. The relative luciferase activities were then converted into logarithm values (binary logarithm, i.e., log2).

The cutoff value set for positives is 0.5849, which indicates that a compound can decrease the luciferase activity to at least 66.7% of the DMSO group.

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