Fig. 9. Stretch-conditioned medium activates p38 via TACE-released TNF-. (A) Stretch-conditioned medium activates p38. C2C12 myoblasts were stretched for 2 hours. The medium in the stretched myoblast culture was collected, cleared of floating cells by centrifugation, and transferred into plates that contained unstretched C2C12 myoblasts for a 15 or 30 minutes incubation. Medium transferred from unstretched myoblasts was used as control. Activation of p38 in the myoblasts was then evaluated by western blot analysis. (B) TACE activity is responsible for p38 activation by stretch-conditioned medium. C2C12 myoblasts were preincubated with or without TAPI (5 µM) or an equal volume of DMSO (1 µl/ml) and stretched for 2 hours. The media were then transferred into plates that contained unstretched C2C12 myoblast cultures and incubated for 15 minutes. Western blot analysis was carried out to evaluate p38 activation. (C) TNF- released into stretch-conditioned medium is responsible for p38 activation. C2C12 myoblasts were stretched for 2 hours in the presence of TNF--neutralizing antibody or pre-immune IgG (5 µg/ml). The media were then transferred to unstretched C2C12 myoblast cultures and incubated for 15 minutes. Western blot analysis was carried out to evaluate p38 activation. (D) Stretch-conditioned medium activates p38 via TNF- receptor-mediated signaling. Culture medium from C2C12 myoblasts that were stretched for 2 hours was transferred to WT or p55^–/– p75^–/– (KO) primary myoblasts and incubated for 15 minutes. Activation of p38 in the myoblasts was then evaluated by western blot analysis