Western blot analysis of full-length and soluble JAM-A. (A) Schematic representation of the domain structure of transmembrane JAM-A and its potential cleavage site at the cell membrane. (B) HUVECs and HEK293 cells were cultured for 8 hours in the absence of fetal calf serum. Cell lysates and conditioned media were then examined for the presence of full-length (fl) and soluble (s) JAM-A by SDS-polyacrylamide gel electrophoresis under reducing conditions and subsequent Western blotting using a monoclonal antibody directed against the second IgG domain of JAM-A. (C) Cell lysates and conditioned media from HUVECs were treated with 2 U/mL N-Glycosidase F for 2 hours at 37°C, and subsequently fl and sJAM-A were analyzed by Western blotting. Data shown are representative of at least 3 independent experiments.