Fig. 4. D-basigin partially colocalizes with actin and integrin. (A-F) High Five cells expressing D-basigin with a C-terminal V5 tag. (A and C) Cells labeled with anti-V5 antibody and visualized with Alexa 488-conjugated secondary antibody. (B,D-F) Cells labeled with Alexa 568-phalloidin to visualize F-actin. (A) D-basigin is expressed diffusely and in numerous vesicles within the cell. There is strong D-basigin expression at cell-cell contacts (arrow). (B) Actin colocalizes with D-basigin at cell-cell contacts (arrow). (C,D) High magnification view of the edge of a basigin-expressing cell (ca. 15 µm total width). There is significant colocalization of D-basigin (C) and actin filaments (D) as shown for marked filaments (asterisks). Some actin filaments do not colocalize with D-basigin (red arrowhead in D). D-basigin-mediated cell spreading requires integrin activity. (E) D-basigin-expressing High Five cells cultured under normal conditions. (F) Cells cultured in the presence of 200 µg/ml GRGDS peptide which competes for integrin binding sites. (G) S2 cell expressing Drosophila PS1ßPS integrins labeled with antibody against D-basigin and visualized with Alexa 568-conjugated secondary. (H) The same cell as in G labeled with a mixture of antibodies to PS1 and ßPS integrin and visualized with Alexa 488-conjugated secondary antibody. D-basigin and integrin colocalize at many sites. Bar, 20 µm (A,B); 60 µm (E,F); 10 µm (G,H).