Fig. 1.

Mechanical stretch activates TACE through Src-mediated phosphorylation in C2C12 myoblasts. C2C12 myoblasts were statically stretched for the indicated time period using the Flexcell® FX-5000™ Tension System. Cell lysate was prepared and subjected to western blotting or immunoprecipitation analysis. (A) Mechanical stretch activates Src. Antibodies specific for Src or Src that is phosphorylated at the Tyr416 residue (pY416) were used in western blot analysis to assess Src activity. (B) Mechanical stretch activates tyrosine phosphorylation of TACE. TACE was immunoprecipitated from cell lysate and analyzed for tyrosine phosphorylation by western blot analysis using antibodies against phosphorylated tyrosine (pTyr) or TACE. (C) The SFK inhibitor PP2 blocks mechanical activation of TACE phosphorylation. C2C12 myoblasts were stretched for 30 minutes with or without pretreatment with 10 µM PP2 or PP3. TACE was immunoprecipitated from cell lysate and analyzed by western blotting with antibodies against phosphorylated tyrosine or TACE. (D) PP2 blocks mechanical activation of TACE. C2C12 myoblasts were stretched for 30 minutes with or without pretreatment with 10 µM PP2 or PP3. TACE activity in cell lysates was analyzed by measuring the rate of cleavage of the peptide containing the TACE-specific cleavage site in pro-TNFα. (E) PP2 blocks mechanical activation of myogenic marker p38 MAPK. On the basis of the previously determined time course of stretch-activation of p38 (Zhan et al., 2007), C2C12 myoblasts were stretched for 120 minutes with or without 10 µM PP2 or PP3. Cell lysate was analyzed for p38 activation using western blotting. Western blots were quantified by densitometry. Data (means±s.e.) were analyzed by ANOVA (A,B,C and E) or Student's t-test (D); *P<0.05 compared with the non-stretched control.