Tb antiphospho 4ebp1 thr37 46 antibody

LANCE^® ultra time-resolved fluorescence resonance energy transfer (TR-FRET) assay (Invitrogen, Carlsbad, CA, USA) was used to determine the mTOR kinase activities of all the compounds following the manufacturer’s instructions, with compound GSK2126458 (Selleck, China) as positive control.39 (link) Briefly, mTOR enzyme (0.1 μg/mL, Invitrogen, Carlsbad, CA, USA), ATP (3 μmol/L (μM)), GFP-4EBP1 peptide (0.4 μM), and test compounds were diluted in kinase buffer (50 mM HEPES pH 7.5, 1 mM EGTA, 3 mmol/L (mM) MnCl₂, 10 mM MgCl₂, 2 mM DTT, and 0.01% Tween-20). The reaction was performed in black 384-well proxiplates (Corning, New York, NY, USA) at room temperature for 1 h, then stopped by adding EDTA to 10 mM. Tb-antiphospho-4EBP1 (Thr37/46) antibody (PerkinElmer, Fremont, CA, USA) was added to each well, and the mixture was incubated at room temperature for 30 min. Test compound concentrations were 10,000, 2500, 625, 156.25, 39.06, 9.77, 2.44, 0.61, 0.15, 0.04 and 0.01 nM. The final DMSO concentration was 1%. A Spectramax 190 reader (Molecular Devices, Valley, CA, USA) was used to measure the intensity of the light in TR-FRET mode (excitation 320 nm, emission 665 nm). All compounds were tested twice, and the results were expressed as the average IC₅₀ (inhibitory concentration 50%) of the two experiments.