Eif2α peptide substrate

The inhibition of HRI by the HRI-i was determined using a luminometric HRI activity assay. This method has been previously described to evaluate the activity of other inhibitors against other kinases like GSK3-β [30 (link)]. Briefly, HRI-i was tested at 1 μM (the concentration used in all the experiments) diluted in kinase buffer (containing final concentrations of: 20 mM Tris pH 8, 50 mM KCl, 25 mM MgCl₂ and 1μM phenylmethysulfonil fluoride) and in presence of 5 μM. eIF2α peptide substrate (Santa Cruz Biotechnology), immunoprecipitated HRI from mouse cortex (goat, Santa Cruz Biotechnology) and 10 μM ATP, to a total assay volumen of 50 μl. The enzymatic reaction was stopped after 5 min of incubation at room temperatura by adding 50μL of Kinase Glo (Promega). After 10 min of stabilization, the luminiscence was measured with a Turner Designs Luminometer Model TD20/20.

HRI activity and the inhibitory ability of the HRI-i was determined using a luminometric assay as previously described for other kinases inhibitors^{38 (link)}. For this assay, 1 μM HRI-i diluted in kinase buffer (20 mM Tris pH 8, 50 mM KCl, 25 mM MgCl2 and 1 µM phenylmethysulfonyl fluoride) was added to a reaction mixture composed of 5 μM eIF2α peptide substrate (Santa Cruz Biotechnology), immunoprecipitated HRI from mouse cortex/hippocampus (goat, Santa Cruz Biotechnology) and 10 µM ATP, in a final volume of 50 μl. After 5 min of incubation at room temperature, the enzymatic reaction was stopped by adding 50 µL of Kinase Glo (Promega). After 10 min of stabilization, the luminescence was measured with a Turner Designs Luminometer model TD20/20. The luciferase signal was proportional to the amount of ATP, and the data are expressed as the inverse of the ATP amount to determine the fold kinase activity.