Figure 4

ET Inhibition of TEM in the Presence of PKA Inhibitors. (A) HMVEC-Ls were preincubated in the presence (+) or absence (-) of H-89 (10 μM) or KT-5720 (10 μM), respectively, before being treated with ET (1000 ng/mL:1000 ng/mL) for 6 h and lysed. The lysates were processed for pCREB immunoblotting. To control for protein loading and transfer, blots were stripped and reprobed for β-tubulin. IB, immunoblot, IB*, immunoblot after stripping. (B) The pCREB signals in each blot described in (A) were quantified by densitometry of pCREB and normalized to β-tubulin signal in the same lane in the same blot. (C) HMVEC-Ls cultured to confluence in assay chambers were pretreated with medium, H-89 (10 μM) or KT-5720 (10 μM), after which they were treated for 4 h with medium, ET, ET with H-89, or ET with KT-5720. The HMVEC-L monolayers were then inserted into wells containing either medium or IL-8 (10 ng/mL), after which calcein-AM-labeled PMNs were added to the upper compartment of each chamber. After 2 h, the contents of each lower compartment were fluorometrically assayed. Each vertical bar represents mean (+/- SEM) TEM of PMNs (%). The n for each group is indicated in each bar. * indicates significantly increased compared to the simultaneous medium only controls at p < 0.05. ** indicates significantly decreased compared to IL-8 alone at p < 0.05.