Figure 4

Immunolabelling of endogenous and exogenous Netrin-4 in VSMC. Cells were fixed and stained with an antibody against Netrin-4. (A) Immunolabelling of endogenous Netrin-4 within the cytoplasm of VSMC. (B) VSMC were incubated with exogenous Netrin-4 for 2 hours. Netrin-4 immunoreactivity is at the cell surface suggesting that the Netrin-4 is bound to the Netrin receptors. Scale bar: 100 μm. (C & D) Cells were transfected with empty vector (pcDN3.1) or with Netrin-4 expressing plasmid (pcDN3.1-N4). Two days after transfection, conditioned media were collected and cells were fixed. As observed in C, transfected cells express Netrin-4. Scale bar: 200 μm. (D) Detection of the protein in the conditioned media by ELISA showing that Netrin-4 is secreted by VSMC.