Fig. 6. Signaling through JAM-A, PI 3-kinase and PKC is required for JAM-A-induced migration. (A) Western blot of protein lysate (50 µg) from mock-, JAM-A- and JAM-A -257-transfected HUVECs, indicating level of expression of JAM-A. [Note: deletion of the cytoplasmic domain (-257) results in a lower-sized band.] (B) Cell migration on vitronectin using cells as in A. (C) Western blot showing the absence of HA-tagged JAM-A -257 in the immunoprecipitate (IP) of anti-_vß₃ from total cell lysate (Input) of serum-starved HUVECs transfected with the JAM-A -257 construct and treated with or without RGDS as indicated. Isotype-specific IgG was used as a negative control (cIgG), and the blot was reprobed with anti-ß3 antibody, to ensure equal loading. Mock-transfected or JAM-A-overexpressing cells were pre-treated with various concentrations of anti-JAM-A antibody (D), or with DMSO or wortmannin or Bis (E) for 20 minutes and then assayed for migration on vitronectin. *P<0.05.