Activation of RhoGTPases following ICAM-1 cross-linking on bEndI1/2.1 re-expressing ICAM-1 wild type or its mutants. Endothelial cells were cross-linked with antimouse ICAM-1 followed by goat antirat IgG (RAM) for 15 minutes. Cross-linked cells were lysed and activated Rho proteins (guanosine triphosphate [GTP]-bound form) precipitated with GST-rhotekin. Total Rho proteins and GST-rhotekin-precipitated Rho proteins were subsequently [32P]ADP-ribosylated, resolved on 15% SDS-PAGE, and exposed to autoradiographic film. L indicates lysate; rh, precipitated GST-rhotekin.