Fig. 12. Expression of a GFP fusion protein containing the VCA domain of N-WASP interferes with EC vacuole formation. (A) ECs were induced to express GFP control or GFP-VCA (a C-terminal domain of N-WASP that activates the Arp 2/3 complex). Extracts were made and samples were run on SDS-PAGE, blotted to PVDF membranes and probed with anti-GFP antibodies. (B) Quantitation of the effects of GFP-VCA chimera on EC vacuole formation at 24 hours of EC morphogenesis. (C) ECs expressing GFP-VCA were cultured in collagen matrices and photographs were taken after 24 hours of culture. Images show ECs in three-dimensional collagen expressing GFP-VCA that are unable to form lumenal structures but form numerous processes. Bar=25 µm.