ERG and RhoJ siRNA knock-down markedly inhibit Rac-1 and Cdc42 Rho GTPase activation, whereas RhoA activation is strongly increased during EC lumen formation in 3D collagen matrices. (A) ECs were infected with recombinant adenoviruses carrying S-epitope–tagged GFP-Cdc42, JamB, and MT1-MMP or GFP as a control. EC cultures in 3D matrices were prepared and after 16 hours, lysates were prepared and incubated with S-protein beads. Beads were eluted with SDS-PAGE sample buffer and samples were run on gels and Western blots probed with antibody against RhoJ. (B) Cultures were established as described in “Methods,” and after 16 hours, detergent lysates were prepared to assess the degree of Cdc42, Rac1, and RhoA activation. Starting material lysates and the eluates from the Pak and Rhotekin beads were assessed by Western blots using anti-Cdc42, anti-Rac1, and anti-RhoA antibodies. Representative images are shown (additional ones are included in supplemental Figure 5B). Quantitation was done by densitometric analysis (n = 3). *P < .01. (C) ECs were treated with the indicated siRNAs (40 nmol/L) for ERG and RhoJ, and 24 hours later, were seeded within 3D collagen matrices to undergo morphogenesis. After 24 hours of culture, EC lysates were prepared and analyzed for the indicated molecules. In each case, we analyzed for phosphorylated kinases as an indicator for kinase activation, as well as the total levels of each kinase.