FIGURE 2.

Influence of Cys³⁹² on the proteolytic activity of hADAMDEC1. A, α2M-cross-linking assay with hADAMDEC1 variants and mWT visualized by Western blot analysis using an anti-HPC4-tag mAb against a N-terminal HPC4 tag on the ADAMDEC1 variants. The different α2M·ADAMDEC1 complexes (I) are observed at the top of the blots, whereas the added protease (III) is seen at the bottom. Also, cross-reactivity of antibodies with an impurity in the α2M preparation is observed (II). B, proteolytic activity against soluble, azo-labeled casein. The sample means are shown by thick horizontal lines. For one-way analysis of variance: ***, p < 0.0001; ns, not significant relative to mock. Student's t test comparing hWT and hC392S is shown with a horizontal bracket. C, proteolytic assay using Cm-Tf (marked by a black dot) as substrate for the indicated hADAMDEC1 variants (500 nm). Samples were analyzed by reducing SDS-PAGE, and primary product bands are marked by black arrows.