Activation of ERK by mechanical stretch and effect of APC. (a) Time-course of extracellular-regulated kinase 1/2 (ERK) activation by alveolar distention. Immunoblotting detection of phosphorylated (activated) ERK-1 (top band) and ERK-2 (bottom band) in reference to actin in mouse whole lung lysates following ventilation with tidal volume of 25 mL/Kg. ERK activation in lung tissue occurs rapidly, within 30 minutes and increases after 60 minutes of injurious ventilation. A representative blot of three experiments and band density analysis of cumulative results in reference to actin are presented. (b) Activated protein C (APC) reduces ERK activation in lung homogenate following ventilator-induced lung injury (VILI). Detection of activated ERK in mouse whole lung lysates by immunoblotting with antibodies binding specifically to phosphorylated ERK. ERK phosphorylation status after four hours of HVt ventilation remains higher in comparison with control mice. APC inhalation significantly diminishes ERK activation. A representative blot and band density analysis of cumulative results in reference to tubulin are presented. (c) Immunohistochemical staining for activated ERK in lung tissue. Phospho-ERK-positive cells stain brown. VILI induces activation of ERK in alveolar cells, which can be prevented by APC. (data expressed as mean ± standard deviation; * denotes P < 0.05 for comparison with LVt-30 min; One-way analysis of variance with post-hoc analysis by Dunnett's test).