Figure 6.

Effect of H-89 and PKIα on 8-Br-cAMP- and PGE₂-induced GM-CSF release from and CREB phosphorylation in HASM cells. Adherent cells were pretreated with H-89 (10 μM; 30 min) or infected with Ad5.CMV.Null or Ad5.CMV.PKIα (MOI=100; 48 h). 8-Br-cAMP (1 mM) or PGE₂ (10 μM) were then added for 30 min. At this point, cells were either processed immediately for CREB phosphorylation by western blotting (a) or exposed to IL-1β (100 pg ml⁻¹) for 24 h to promote GM-CSF release, which was measured by ELISA (b and c). Each bar represents the mean±s.e.m. of four independent determinations using tissue from different donors. Indomethacin (10 μM) was present throughout the experiment. See Methods for further details. ^*P<0.05, significant inhibition of IL-1β-stimulated GM-CSF release. (Key: lanes 1 & 2, Naïve; lanes 3 & 4, Ad5.CMV.Null; lanes 5 & 6, Ad5.CMV.PKIα, lanes 7 & 8, H-89).