Dual Specificity Phosphatase 1 Regulates Human Inducible Nitric
Oxide Synthase Expression by p38 MAP Kinase
Phosphorylation of p38 MAPK and its substrate MK2 in response to stimulation with cytokine mixture or LPS in A549 and J774 cells. (a) A549 and J774 cells were stimulated with the cytokine mixture (CM: TNF, IFNγ, and IL-1β; 10 ng/mL each) or LPS (10 ng/mL), respectively for the time indicated. Cells were then harvested for protein extraction, and phosphorylation of p38 MAPK was detected by Western blot. The gel is a representative of six separate experiments with similar results. Chemiluminescent signal was quantified, and phosphorylated p38 MAPK was normalized against total p38 MAPK. Phosphorylation levels are expressed in arbitrary units, unstimulated cells set as 1, and the other values are related to that. Results are expressed as mean ± S.E.M.; . One-way ANOVA with Dunnett’s posttest was performed, and statistical significance was indicated with compared with unstimulated cells. ((b) and (c)) The effect of SB202190 and BIRB 796 on the phosphorylation of MK2 in response to cytokine mixture in A549 and J774 cells. Cells were preincubated with SB202190 (1 μM) or BIRB 796 (100 nM) for 30 min and stimulated with cytokine mixture (A549 cells) or LPS (J774 cells) for 30 min, and the phosphorylation of MK2 was detected by Western blot. The gels are representatives of six separate experiments with similar results.