Blockade of PDGF-BB and HB-EGF, using neutralizing antibodies or soluble protein fragments, leads to abrogated EC-induced pericyte motility responses in 3D collagen matrices. Nuc-pericytes were allowed to coassemble with ECs either in the presence or absence of neutralizing antibodies to PDGF-BB and HB-EGF or soluble protein fragments to PDGFRβ and the HB-EGF receptor, ErbB4. Real-time video analysis was carried out to determine the effect of these molecules on pericyte motility. (A) The movement of nuc-pericytes was tracked, using MetaMorph software, with measures of pericyte average velocity, average total distance of movement, and average distance from the origin shown. As demonstrated, disruption of PDGF-BB and HB-EGF signaling in combination leads to blockade of each of the measurements made as markers of pericyte motility in the presence of ECs. (B) Representative images of the nuc-pericyte tracking analysis are shown to demonstrate the inability of pericytes to migrate under conditions of combined PDGF-BB and HB-EGF inhibition. n ≥ 5; P ≤ .01.