Confluent HUVECs show cytoplasmic but not membrane immunofluorescent staining for CADM1 with chicken monoclonal anti-CADM1. In contrast, migrating HUVECs show both cytoplasmic and membrane staining for CADM1 in lamellopodia and filopodia at the leading edge of cell migration. (A) Confluent HUVECs stained for CADM1 (green) and CD31 (red). CD31 clearly demarcates the intercellular boundaries. (B) Confluent HUVECs stained only for CADM1 show no evidence of membrane staining at the intercellular boundaries. (C) Migrating HUVECs show CADM1 staining in the cytoplasm and at the leading edge of lamellopodial membranes (white arrows). (D) The image in panel C counterstained with Phalloidin (red) shows colocalization of actin with CADM1 (combination appears yellow) at the leading edge of lamellopodial membranes (white arrows). (E) Migrating HUVECs stained for CADM1 (green) and CD31 (red) show CADM1 staining of filopodia colocalized at the membrane surface with CD31. CADM1 staining is present along the walls and at the leading edge of the filopodia. White arrows denote filopodia and boxed area a lamellopodia. (F) The boxed area of panel E at greater magnification shows the presence of localized high-intensity staining of CADM1 at the leading edge of a lamellopodia, some of which appears to be associated with developing filopodia.