Fig. 5. Integrin _vß₃ associates with JAM-A. (A) Western blot showing the precence of JAM-A in the immunoprecipitate (IP) of anti-_vß₃ from total cell lysate (Input) of serum-starved untransfected HUVECs treated with or without RGDS as indicated. Isotype-specific IgG (cIgG) was used as a negative control, and the blot was reprobed with anti-ß3 antibody, to ensure equal loading. (B) Immunofluorescence images of live, adherent HUVECs incubated with FITC-XT199. (i-vi) XT199 accumulation at the cell-cell junction was monitored by confocal microscopy from time 0-20 minutes. Arrowheads indicate the position of the cell-cell junctions. (v) FITC-XT199 accumulation at the cell-cell junction of live HUVECs was ablated as a result of competitive inhibition by the addition of excess 1 mM RGDS. (vi) Live HUVECs were pre-treated with 100 µM RGDS compound for 20 minutes to allow integrin _vß₃ accumulation at the cell-cell junction. The live cells were then fixed and treated with anti-_vß₃ antibody to ensure the accumulation was _vß₃ specific (arrowhead). Data shown are representative of three separate experiments. Bar, 10 µm.