HNE inhibition of TrxR activity inversely correlates with MKK3/6 and p38 MAPK activation. (A-B) THP-1 cells were treated with varying concentrations of HNE for 1 hour (A) or with 20 µM HNE for varying time periods (B). After treatment, cells were washed, resuspended in the assay buffer, and sonicated to lyse cells. Equal amounts of protein were taken to measure TrxR activity using the TrxR activity assay kit. (C) Cell lysates of THP-1 cells treated with 20 µM HNE for varying time periods were probed to analyze the phosphorylation status of MKK3/6 and p38 MAPK by western blot analysis. (D) The phosphorylation status of MKK3/6 and p38 MAPK upon HNE stimulation was quantified and plotted against HNE-induced inhibition of TrxR activity at identical time periods. (E) THP-1 cells were treated with the antioxidant NAC (3 mM) or the electron chain transport inhibitors antimycin (Anti; 10 µM) or oligomycin (Oligo; 5 µM) for 1 hour prior to HNE treatment for 4 hours. TrxR activity was analyzed as described above. ***P < .001. ns, not significant; TNB, 5-thio-2-nitrobenzoic acid.