Examined no matter if UA could protect MKP-1 protein expression and activity in metabolically stressed THP-1 monocytes. At three mM, UA prevented the metabolic stress-induced degradation of MPK-1 (Fig. 3A and B) and totally rescued MKP-1 activity in metabolically NLRP3 Agonist drug primed THP-1 monocytes (Fig. 3C). Loss of MKP-1 activity leads to the PPARγ Inhibitor MedChemExpress hyperactivation of p38, as measured by the phosphorylation of p38, both in resting THP-1 monocytes and in response to MCP-1 stimulation . We thus determined if UA also prevents the hyperactivation of p38 in metabolically primed THP-1 monocytes. UA normalized p38 phosphorylation to levels found in wholesome manage cells (Fig. 3D). These information suggest that, below conditions of metabolic strain, UA protects MAPK signaling pathways that manage monocyte adhesion and migration, by stopping MKP-1-S-glutathionylation, inactivation and degradation.S.L. Ullevig et al. / Redox Biology two (2014) 259Fig. 2. UA reduces actin- and total-S-glutathionylation induced by metabolic stress. THP-1 monocytes in RPMI 1640 medium (5 mM glucose, 10 FBS) were treated with 0.three, 1, 3, 10 mM UA or vehicle. HG (20 mM glucose) plus native LDL (one hundred mg/ml) was present for 20 h exactly where indicated. Cells were lysed within the lysis buffer containing ten mM NEM. Actin- and protein-S-glutathionylation was assessed by Western blot evaluation working with the anti-glutathione antibody. Western Blot information for actin-S-glutathionylation is summarized in a . (A) A representative Western Blot is shown. (B) Quantitation by Western blot evaluation assessed applying an anti-glutathione antibody is shown of actin-Sglutathionylation in response to escalating doses of UA. n4, mean7 SE. # versus one hundred actin-S-glutathionylation, P .004 (1 mM), P .003 (3 mM), Pr 0.001 (10 mM). (C) Quantitative information for actin-S-glutathionylation plus the effects of 3 mM UA. Information is represented as fold transform induced by HGLDL (red bar) and HGLDL3 mM UA (green bar) versus unprimed control cells (white bar). n3, imply 7 SE; nversus Manage, P0.006, # versus HGLDL, P0.022. (D) Total protein-S-glutathionylation was determined by Western blot along with the density with the complete lane was measured and normalized to actin. Total protein-S-glutathionylation is represented as fold adjust induced by HG �LDL (red bar) and HGLDL mM UA (green bar) versus unprimed control cells (white bar). n, mean7 SE, nversus manage, Po 0.001, #versus HG �LDL, P0.003.Fig. 3. UA rescues MKP-1 protein expression and activity in metabolically primed THP-1 monocytes. THP-1 cells were treated for 20 h with 3 mM UA or vehicle manage inside the presence of HGLDL. (A) Representative Western blot MKP-1 protein levels. (B) Quantitation of Western blot evaluation. Information was normalized to actin and is shown as mean7SE of 3 independent experiments. nversus unprimed manage cells (no metabolic tension), P.017; #versus HGLDL primed cells, P.012. (C) MKP-1 phosphatase activity was assessed applying a modification on the commercially offered Malachite Green-based PTP assay as described under Material and Approaches. n, nversus unprimed manage cells (no metabolic strain), P0.002; #versus HGLDL, Po0.001. (D) Phospho p38 was measured by Western blot evaluation as described in “Material and methods” section. Information was normalized to total p38. n3, mean7SE; nversus unprimed handle cells (open bar), P.003, #versus HGLDL (red bar), Po0.001, HG�LDL mM UA (green bar).S.L. Ullevig et al. / Redox Biology 2 (2014) 259Fig. 4. UA prevents Nox4 protein induction by metabolic pressure. (A).