Figure 6.

Fibulin-5 loses its elastogenic activity upon proteolytic cleavage. (A–E) Human skin fibroblasts were cultured in serum-free media without addition of proteins (A), with wild-type fibulin-5 (B), with the cleaved form of fibulin-5 (C), or with wild-type fibulin-5 and 500 μM BAPN (D). Each FLAG-tagged recombinant protein was added to the culture at a final concentration of 4 μg/ml. Cultures were stained with anti-FLAG antibody (top) and anti–human elastin antibody (middle). The bottom images were produced by superimposition of the top and middle images, together with DAPI nuclear staining. Bars, 50 μm. (E) Quantitation of insoluble (i.e., cross-linked and mature) elastin produced by cells cultured with various amounts of wild-type and cleaved fibulin-5 added to the medium. Cultured skin fibroblasts were metabolically labeled with [³H]valine during the culture period, and the radioactivity of the NaOH-insoluble fractions was quantitated. The radioactivity count was corrected by the relative cell number of separate wells measured with a modified MTT assay, although neither wild-type nor cleaved fibulin-5 significantly affected the cell number (not depicted). Data were obtained as quadruplicates, and the mean ± the SD is shown.