Figure 5.

IL-27 down-regulates SPTBN1 through a TAK-1–mediated p38 MAPK signaling pathway. On day 4 during differentiation, macrophages were stimulated with IL-27 (50 ng/ml), with or without the pretreatment of a JAK inhibitor or its solvent control DMSO. (A) Whole-cell lysates were harvested 15 min after IL-27 stimulation, and the phosphorylation of STAT1, STAT2, and STAT3 was examined by Western blotting using specific antibodies against p-STATs (Cell Signaling Technology). IFN-α–treated macrophage lysate was included to validate the antibody against phosphorylated STAT2. (B) Macrophages were pretreated with a JAK inhibitor (EMD), a p38 MAPK inhibitor (Cell Signaling Technology), or the solvent control DMSO. After 2 h, macrophages were stimulated with IL-27 (50 ng/ml). Gene expression of SPTBN1 was examined by RT-PCR 24h after IL-27 stimulation. Data shown represent means ± SE of three independent experiments. (C) Macrophages were pretreated with a TAK-1 inhibitor (Cell Signaling Technology) or mock treated for 2 h. Cells were then stimulated with IL-27 (50 ng/ml) and harvested at different time points as indicated. The phosphorylation of p38 MAPK was examined by Western blotting using an anti–p-p38 antibody (Cell Signaling Technology). The expression of unphosphorylated p38 was shown as an internal control. (D) Macrophages were pretreated with a TAK-1 inhibitor (Cell Signaling Technology) or mock treated. After 2 h, macrophages were stimulated with IL-27 (50 ng/ml). Gene expression of SPTBN1 was examined by RT-PCR 24h after IL-27 stimulation. Data shown represent means ± SE of three independent experiments. (E) Recombinant human IL-27 #1 and IL-27 #2 were obtained from R&D Systems and HumanZyme, respectively. On day 4 during differentiation, macrophages were stimulated with IL-27 #1 and IL-27 #2 (50 ng/ml). Gene expression of SPTBN1 was examined by RT-PCR 24 h after IL-27 stimulation. Data shown represent means ± SD of three independent samples.