Effect of tryptase ϵ on human bronchial smooth-muscle cells. (A) Human bronchial smooth muscle cells were placed in serum-free medium alone (control) or with tryptase ϵ, its C90A mutant, or its C90A/D92E mutant. After an overnight incubation, the conditioned medium was collected, concentrated, and subjected to SDS-PAGE under nonreducing conditions. The resulting protein blots were probed with anti-uPA (left), anti–PAI-1 (middle), and anti-uPAR (right) antibodies. Not shown are the immunoblot data of the residual cell lysates, which in all instances contained substantial amounts of uPAR. Although some uPAR is shed in tryptase ϵ–treated cells, most of the receptor remains cell associated. (B) Samples of the conditioned medium also were evaluated for the presence of enzymatically active uPA using the substrate H-Glu-Gly-Arg-pNA.