Figure 5. ERK MAPK is required for flavone induced SOCS3 expression, but not for inhibition of STAT3 phosphorylation.

(a) COS-1 cells were transfected with a luciferase reporter construct encoding the minimal murine Socs3 promoter [24]. In addition, cells were co-transfected with Renilla luciferase vector to normalize luciferase activity and to correct for transfection efficiency. Cells were then stimulated overnight with either 100 μM flavone or 100 nM PMA, in the presence of 1 μM of the MEK inhibitors PD184352 or AZD6244. Cell extracts were prepared and luciferase activities from three separate experiments were quantified. Significant increases in luciferase activity relative to diluent-treated cells are indicated [***P<0.001 (n=3)]. Significant reductions in luciferase activity relative to stimulated cells are also indicated (###P<0.001). (b) HUVECs pre-incubated with either PD184352 or AZD6244 and then stimulated with IL-6 (5 ng/ml) plus IL-6Rα (25 ng/ml), flavone or trans-resveratrol. Cell extracts were then immunoblotted with antibodies against SOCS3, and total and phosphorylated forms of ERK and STAT3 as indicated.