Up-regulation of fibronectin, nidogen-1, laminin isoforms, and perlecan occurs selectively in EC-pericyte cocultures to regulate EC basement membrane assembly. ECs alone, pericytes alone, or both ECs and pericytes were seeded randomly in 3D collagen matrices and allowed to assemble into tube networks. These networks were then either isolated for RNA or lysed for Western blot analysis. (A) mRNA expression of basement membrane components (using species-specific primer sets to HUVECs versus bovine pericytes) was examined at 1, 3, and 5 days of culture. ECs and pericytes alone in culture were examined as well as EC-pericyte cocultures. Relative expression patterns are shown, with glyceraldehyde-3-phosphate dehydrogenase (G3PDH) as a control. (B) Western blot analysis of collagen IV, laminin isoforms, nidogen 1/2, and fibronectin are shown demonstrating protein levels in EC-only (HUVECs), EC-pericyte coculture (HUVECs, bovine pericytes), and pericyte-only cultures (bovine pericytes). CM refers to conditioned medium; L refers to lysates. Actin blots are included as a loading control.