Fig. 6

In vivo contribution of NE to degradation of endogenous E-cad. a, Representative image of equal BAL fluid protein aliquots (in 20 μl) from control mice and mice i.n. challenged with P. aeruginosa for 4 h or O.N. that were reduced and processed for immunoblotting using E-cad N-terminal antibody. Both 4 and O.N. time points revealed a gradual degradation of endogenous E-cad with generation of a distinct ~80 kDa cleavage fragment that migrated similar to the cleavage fragment generated by purified NE. Note cell-free NE−/− BAL fluids showed less cleaved E-cad fragment by comparison to cell-free WT BALs. b, Densitometric analysis of immunoblot images corresponding to all cell-free BAL fluids per group, genotype and condition confirmed the protein profile of Fig. a. c, enzymatic activity assay analysis revealed the presence of active NE in cell-free WT, but not NE−/−, BAL fluids that became abundant over time (O.N.). Data were similar in all challenged mice and mouse experiment was repeated once with reproducible findings (bars ± SEM; **** corresponds to p < 0.0001for infection versus PBS)