FIG. 5.

Matrix binding domain of fibulin-5. A. Cell-based matrix binding assays. Confluent FBCs were incubated with conditioned media containing F5-V5 or ΔC-V5 in the absence of serum for 24 h, followed by immunostaining with anti-V5 or antielastin (PR396) antibodies. Double staining of the FBC matrix shows partial colocalization of fibulin-5 and elastin. Incubation with ΔC-V5 and staining for V5 served as a negative control. B. FBCs were incubated with F5-V5 or Δ1-V5 and stained with DAPI, anti-V5, anti-fibrillin-1, and antielastin antibodies. Δ1-V5 showed a severe reduction in matrix binding but did not affect endogenous elastin or fibrillin-1 matrix formation. Protein content in the media after 24 h of incubation with FBC cells was evaluated by Western blot analysis (right). C. FBCs were incubated with ΔC-V5, F5-V5, ΔH-V5, or a 1:1 ratio of F5-V5 and Δ1-V5 in the absence of serum for 24 h and stained with DAPI and anti-V5 antibody. The ΔH mutant showed a fibrillar matrix pattern. Coincubation of F5-V5 with Δ1-V5 did not affect matrix binding of the wild-type fibulin-5.