FIG. 4.

Time courses of Rho, Rac and CDC42 activation in CNF-stimulated endothelial cells. HUVEC were incubated with CNF (2 μg/ml) for the indicated time periods and then subjected to Rhotekin RBD pull down to detect active, GTP-loaded RhoA (A) or PAK-CRIB pull down to detect active, GTP-loaded Rac1 (B) or CDC42Hs (C). The input in A, B, and C represents approximately 5% of the cell lysate used for the respective pull down at the indicated time point. Immunoblots of pull down assays and inputs were investigated with the same antibodies. RhoA and Rac 1 were activated at 3, 6, and 24 h, whereas CDC42Hs was already active at 0 h. The level of Rac protein but not of Rho or CDC42Hs decreased during CNF stimulation. One representative experiment out of three similar ones is shown.