Ang-1 enhances IL-8 mRNA stability through Erk1/2 activation. (A) Cells were first exposed for 1 hour to Ang-1 and were then maintained in fresh medium, medium containing 5 μg/mL ActD (− Ang-1 + ActD), or ActD and Ang-1 (300 ng/mL; + Ang-1 + ActD). Cells were then collected after different time periods. Total RNA was then extracted, and IL-8 and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA (controls) levels were detected with Northern blotting. (B) Means (± SEM; n = 3) of IL-8 mRNA intensities measured in cells undergoing protocols shown in panel A. mRNA intensities expressed as percentage of those measured after 1 hour of Ang-1 treatment. *P < .05 compared with −Ang-1 plus ActD. (C,D) Representative Northern blot and means (n = 3) of IL-8 mRNA. Cells were treated with Ang-1 for 1 hour and were then maintained in medium containing ActD plus Ang-1 (+ Ang-1 + ActD) and Ang-1 plus ActD + PD98059 (30 μM). Cells were then collected after different time periods, and total RNA was extracted and underwent Northern blotting for IL-8 and 18S levels. (E) Mean values (± SEM; n = 3) of IL-8 mRNA measured in cells which were treated first with Ang-1 for 1 hour (100% values) and were then maintained for an additional 1 hour in media containing Ang-1 plus ActD, Ang-1 plus AcD plus WM (50 nM), Ang-1 plus ActD plus LY294002 (10 μM), and Ang-1 plus ActD plus SP600125 (15 μM).