Fig. 7. Pak2 and Pak4 are strongly activated during EC lumen and tube formation. ECs were suspended in collagen matrices and were allowed to undergo EC tube morphogenesis for 48 hours for time-lapse analysis or western blot analysis. (A) The areas of EC lumens (>10 lumens/field) were traced using Metamorph software from digital images of the indicated time-points and plotted (from two representative movies). (B) Extracts of EC cultures in 3D collagen matrices were prepared at the indicated time-points and probed for phospho-Pak2, Pak2, phospho-Pak4 and Pak4. Beta-actin was used as a loading control. (C) The relative levels of phosphorylated Pak2 or Pak4 and total Pak2 or Pak4 were quantified by using Scion imaging software. The data are expressed in arbitrary units (AU)±s.d. (n=2).