EB1, p150^Glued, and Clasp1 control vascular tube morphogenesis by affecting a lumen signaling kinase cascade and tubulin posttranslational modifications, including induced acetylation and detyrosination events. (A) Time course experiment to monitor the influence of siRNA-treated control, EB1, p150^Glued, and Clasp1 ECs during lumen and tube formation after the indicated times of culture. EC luminal areas were traced and analyzed using Metamorph software. Data are shown as mean EC luminal area ± standard deviation (n = 3). (B) EB1, p150^Glued, and Clasp1 control EC morphogenesis in 3D collagen matrices through posttranslationally modified tubulins. EC lysates were harvested at 24 hours for immunoblot analysis and probed with indicated antibodies. Depletion of EB1, p150^Glued, and Clasp1 by siRNA interference markedly decreases posttranslationally modified tubulin expression (compared with total α -tubulin) that accompanies blockade of EC lumen and tube formation in 3D collagen matrices. (C) ECs in 3D collagen matrices were lysed at the indicated time points for immunoblot analysis to analyze the expression or presence of EB1, p150^Glued, Clasp1, APC, SIRT2, HDAC6, polyglutamylated tubulin, detyrosinated-tubulin, delta-2-tubulin, acetylated tubulin, tyrosinated tubulin, α-tubulin, and γ-tubulin during EC tube formation over time. An actin immunoblot was used as a loading control. (D) Depletion of EB1, p150^Glued, or Clasp1 by siRNA interference impairs an EC lumen kinase-signaling cascade in 3D collagen matrices. Phospho-Pak2, -Pak4, -B-Raf, and -Erk are dramatically inhibited during EC lumen and tube formation in EB1, p150^Glued, or Clasp1 knockdown cells. Total kinase levels as well as actin levels were determined. These blots were from a representative experiment that was repeated twice. The specificity of siRNAs and their ability to knockdown the protein expression was confirmed by immunoblot analysis.