RvD1 enhances IL-4-induced STAT6 activation. (A) BV-2 cells were treated with IL-4 (10 ng/ml) for the indicated times, expression of phosphorylated STAT6 was assessed by Western blot. Quantification of phospo-STAT6 from three independent experiments indicated that the effect of IL-4 was obvious at 60 minutes. (B) BV-2 cells were treated with RvD1 (100 nM), IL-4 (10 ng/ml), Boc-2 (10 μM) or combination of them. Levels of phosphorylated STAT6 were detected by Western blot 60 minutes after IL-4 stimulation. A representative result from three independent experiments is shown. Quantification of phosphorylated STAT6 was normalized by β-actin. (C) BV-2 cells were treated as in (B). Nuclear extracts were prepared 120 minutes after IL-4 treatment and used to analyze STAT6 DNA binding activity by Electrophoretic Mobility Shift Assay (EMSA). Binding specificity was confirmed by unlabelled probe (200-fold in excess) to compete with labeled oligonucleotide. Results were confirmed by three independent experiments. Data are presented as mean ± SEM for three independent experiments. Asterisks indicate statistically significant difference (*P < 0.05, **P < 0.01).