Efficient and precise detection techniques, along with extensive repertoires of specific binding reagents, will be needed to meet the challenges of proteome analyses. The recently established proximity ligation mechanism enables sensitive high-capacity protein measurements by converting the detection of specific proteins to the analysis of DNA sequences. Proximity probes containing oligonucleotide extensions are designed to bind pairwise to target proteins and to form amplifiable tag sequences by ligation when brought in proximity. In our previous report, both the ligatable arms and the protein binders were DNA molecules. We now generalize the method by providing simple and convenient protocols to convert any polyclonal antibodies or matched pair of monoclonal antibodies to proximity probe sets through the attachment of oligonucleotide sequences. Sufficient reagent for >100,000 proximity ligation assays can be prepared from 1 g of antibody. The technique is applied to measure cytokines in a homogenous test format with femtomolar detection sensitivities in 1-l samples, and we exemplify its utility in situations when only minute sample amounts are available.

During vascular development, endothelial platelet-derived growth factor B (PDGF-B) is critical for pericyte recruitment. Deletion of the conserved C-terminal heparin-binding motif impairs PDGF-BB retention and pericyte recruitment in vivo, suggesting a potential role for heparan sulfate (HS) in PDGF-BB function during vascular development.We studied the participation of HS chains in pericyte recruitment using two mouse models with altered HS biosynthesis. Reduction of N-sulfation due to deficiency in N-deacetylase/ N-sulfotransferase-1 attenuated PDGF-BB binding in vitro, and led to pericyte detachment and delayed pericyte migration in vivo. Reduced N-sulfation also impaired PDGF-BB signaling and directed cell migration, but not proliferation. In contrast, HS from glucuronyl C5-epimerase mutants, which is extensively N-and 6-O-sulfated, but lacks 2-O-sulfated L-iduronic acid residues, retained PDGF-BB in vitro, and pericyte recruitment in vivo was only transiently delayed. These observations were supported by in vitro characterization of the structural features in HS important for PDGF-BB binding. We conclude that pericyte recruitment requires HS with sufficiently extended and appropriately spaced N-sulfated domains to retain PDGF-BB and activate PDGF receptor ␤ (PDGFR␤) signaling, whereas the detailed sequence of monosaccharide and sulfate residues does not appear to be important for this interaction.[Keywords: PDGF-B; angiogenesis; heparan sulfate; pericyte; vascular development] Supplemental material is available at http://www.genesdev.org. Tissue morphogenesis depends on cell-cell interactions, controlling directed cell migration proliferation, differentiation, and cell survival. Specificity is often regulated at the level of selective ligand-receptor interaction. However, the spatial distribution and local concentration of the ligand determine the range of the signal, and, as exemplified by morphogens of the hedgehog, TGF␤, and Wnt family members, also the nature of the signal. Indeed, spatial restriction defines the activities of most peptide growth factors and many secreted neural guidance molecules. In vascular development, peptide growth factors of the VEGF and platelet-derived growth factor (PDGF) families regulate the migration and proliferation of endothelial cells and supporting mural cells; i.e., pericytes (PC) and vascular smooth muscle cells (vSMC). The longitudinal migration and proliferation of vSMC/PC depend on paracrine signaling of endothelial derived PDGF-B to PDGF receptor-␤ (PDGFR␤) expressed on vSMC/PC (Lindahl et al. 1997;Hellström et al. 1999). PDGF-B is secreted as a homodimer (PDGF-BB), which signals by mediating dimerization of its receptor. Conditional inactivation of Pdgf-b in the endothelium demonstrated that endothelial cells are the

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The detection of weakly expressed proteins and protein complexes in biological samples represents a fundamental challenge. We have developed a new proximity-ligation strategy named 3PLA that uses three recognition events for the highly specific and sensitive detection of as little as a hundred molecules of the vascular endothelial growth factor (VEGF), the biomarkers troponin I, and prostate-specific antigen (PSA) alone or in complex with an inhibitor--demonstrating the versatility of 3PLA.

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