Roles of the transcription factors AP-1 in Ang-1–induced IL-8 production. (A) Top: structure of human IL-8 promoter (−133/+44) identifying AP-1 and NFκB-like binding elements. Sequences in brackets delineate in small letters mutations in these binding elements. Bottom: IL-8 promoter activity (normalized luciferase activity) measured in response to vehicle, Ang-1 and Ang-1 plus PD98059 (30 μM), WM (50 nM), and SP600125 (15 μM). *P < .05 compared with vehicle. Error bars represent SEM. (B) Binding of nuclear extracts from vehicle- and Ang-1–treated HUVECs to IL-8 specific AP-1 DNA probe in the absence (−) and presence of antibodies to c-Fos, FosB, c-Jun, JunB, and JunD. Competition with cold and mutated probes was performed as specificity control. (C) Effects of Ang-1 (300 ng/mL) on phosphorylation of c-Fos (Thr325), c-Jun (Ser63 and Ser73), total c-Fos, c-Jun, and JunD proteins. (D) The effect of 1 hour pretreatment with PD98059 (30 μM), SP600125 (15 μM), and WM (50 nM) on c-Jun phosphorylation (Ser63 and Ser73) measured after 15 minutes of Ang-1 (300 ng/mL) exposure.