IL-33 induces Akt/eNOS activation and NO production via ST2/TRAF6. (A-D) HUVECs were pretransfected with ST2 siRNA (40 nmol/L) and TRAF6 siRNA (40 nmol/L) before IL-33 treatment. Cells were harvested for assay at 40 hours after transfection. (A-B) HUVECs were treated with 20 ng/mL IL-33 for 30 minutes. Cells were harvested and phosphorylation of Akt and eNOS by IL-33 were detected by Western blotting. Blots are representative of 3 independent experiments. Densitometric analyses are presented as the relative ratio of P-Akt to Akt and P-eNOS to eNOS. The relative ratio in untreated control is arbitrarily presented as 100 (bottom). (C-D) After transfection, HUVECs were treated with 20 ng/mL IL-33 for 4 hours and incubated with DAF-FM diacetate for 1 hour. The relative levels of intracellular NO were determined from the fluorescence intensity of DAF-FM. Three independent experiments were performed in duplicate. Data are means ± SDs; *P < .01 vs control cell without IL-33; #P < .01 vs control cell with IL-33.