VEGF 165 binding to MS-5 cells. (A) Left: Structure of native VEGF 165 (V165) and its proteolytic fragments (V110 and V55). Right: Confluent MS-5 cells were incubated with 0.5 ng/mL of125I VEGF 165 and various amounts of V165, V110 (domain encoded by exons 1 to 5 that do not bind neuropilin-1), and V55 (domain encoded by exons 7 and 8 that bind neuropilin-1). Nonspecific binding was measured in the presence of 2 μg/mL of unlabeled V165. The competitive displacement is expressed as the average of iodinated V165 specific binding in triplicate assays. Standard errors were less than 10%. (B) Subconfluent cells were washed twice in ice-cold binding medium and incubated with 5 ng/mL 125I-VEGF for 2 hours in the absence (lane 0) or in the presence of 1 μg/mL of V165 (lane V165), V55 (lane V55), or sema III-AP (lane sema III). For MS-5 cells, cross-linked complexes were incubated in the presence of 2 μg/mL of anti–neuropilin-1 antibodies. Proteins were resolved on a 7% SDS polyacrylamide gel, stained with Coomassie Blue, and autoradiographed. Position of molecular-weight markers is indicated on the right. pgs-A745: xylosyltransferase deficient pgs-745 CHO cells constitutively expressing either neuropilin-1 (pgs-A745 NP-1) or KDR (pgs-A745 KDR). The upper band corresponds to species that did not enter the gel. Arrowheads indicate cross-linked complexes.
