The binding reaction was performed for 20 minutes in a volume of 30 l containing 1x binding buffer (20 mM Hepes pH 7.9, 1.4 mM MgCl2, 1 mM ZnSO4, 40 mM KCl, 0.1 mM EDTA, 5% Glycerol), 0.2 g Poly(dI-dC) for DNA EMSA, 2 g of tRNA for RNA EMSA, 0.1 g BSA, 10 mM DTT and 0.1% NP40
Phosphorylation of eIF2 by PERK in response to ER stress decreases translation of mRNA and reduces stress (Harding et al , 2003)
In addition, pro-inflammatory cytokines can act to steal tryptophan away from serotonin and melatonin pathways (via up-regulation of the enzymes IDO and TDO), increasing the activity of the NMDA agonist quinolinic acid, and further antagonising stimulation of NMDA pathways, which subsequently exacerbates mood and sleep issues
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