FIG. 8.

Fibulin-4 interacts with tropoelastin. (A) Purified recombinant mouse fibulin-4. A total of 1 μg of purified fibulin-4 was used for the Coomassie blue-stained gel, and 10 ng of the protein was used for the Western blot. An anti-FLAG antibody was used for immunodetection. A single band with the correct mass for fibulin-4 was detected by both Coomassie blue staining and immunoblotting. (B) Solid-phase binding assay using recombinant fibulin-4 as a soluble ligand. Note that fibulin-4 binds to tropoelastin in the presence of Ca²⁺ but that the binding is inhibited in the presence of EDTA. Data were obtained as the results of triplicate experiments, and values shown are means ± standard deviations. (C) Coimmunoprecipitation of fibulin-4 with tropoelastin. Lanes 1 to 4, antitropoelastin blotting of immunoprecipitates from lysates of 293T cells transfected with both tropoelastin and fibulin-4 (lanes 1 and 3) or tropoelastin alone (lanes 2 and 4). Tropoelastin was detected in the immunoprecipitate by anti-fibulin-4 (7B9) (lane 3). Lanes 5 to 8, anti-fibulin-4 (11E2) blotting of immunoprecipitates from lysates of 293T cells transfected with both tropoelastin and fibulin-4 (lanes 5 and 7) or fibulin-4 alone (lanes 6 and 8). Fibulin-4 was detected in the immunoprecipitate by antitropoelastin (lane 7). IP, immunoprecipitation; TE, tropoelastin; fb4, fibulin-4.