Human Anti-NRP2 Recombinant Antibody (CAT#: PABW-095)

Figure 1 Identification of the NRP1 domain that binds VEGF165.

Plasmids containing NRP1 domains were transfected into PAEC. Top panels, aliquots of CM were collected, incubated with 125I-VEGF165 in solution, and immunoprecipitated with anti-Myc antibody. Immunopreciptates were analyzed by SDS-PAGE and autoradiography. Solid arrows, 125I-VEGF165 monomer; open arrows, 125I-VEGF165 dimer. Bottom panels, aliquots of the same samples were analyzed for individual domain protein content by Western blot using anti-Myc antibody.

Mamluk, R., Gechtman, Z. E., Kutcher, M. E., Gasiunas, N., Gallagher, J., & Klagsbrun, M. (2002). Neuropilin-1 binds vascular endothelial growth factor 165, placenta growth factor-2 and heparin via its b1b2 domain. Journal of Biological Chemistry.

Figure 2 The a1a2 domain enhances the binding of VEGF165, but not PlGF-2 to the b1b2 domain.

Five nanograms of 125I-VEGF165 (A) or 125I-PlGF-2 (B) were incubated with 2 g/ml purified b1b2 or a1a2/b1b2 recombinant proteins and heparin (1 g/ml) in solution, followed by cross-linking with disuccinimidyl substrate, SDS-PAGE, and autoradiography. A, lane 1, no NRP1 domain; lane 2, complexes of 65 and 80 kDa were formed with the b1b2 domain (open arrows); lane 3, complexes of 110–135 and 180–235 kDa were formed with a1a1/b1b2 (solid arrows). B, lane 1, no NRP1 domain; lane 2, a complex of 65 kDa was formed with the b1b2 domain (open arrow); lane 3, a complex of 120 kDa was formed with a1a1/b1b2 (solid arrow).

Mamluk, R., Gechtman, Z. E., Kutcher, M. E., Gasiunas, N., Gallagher, J., & Klagsbrun, M. (2002). Neuropilin-1 binds vascular endothelial growth factor 165, placenta growth factor-2 and heparin via its b1b2 domain. Journal of Biological Chemistry.

Figure 3 Characterization of PlGF-2/NRP1 interactions.

A, immunoprecipitation: 125I-PlGF-2 was incubated in solution without (lane 1) or with 250 ng of pure Myc-tagged NRP1 b1b2 (lane 2) and a1a2/b1b2 (lane 3) domains in the presence of 1 g/ml heparin, followed by immunoprecipitation with anti-Myc antibody. Immunopreciptates were analyzed by SDS-PAGE and autoradiography. B, 125I-PlGF-2 and 125IVEGF165 were incubated in solution without (lanes 1 and 4) or with equal amounts of pure Myc-tagged NRP1 b1b2 domain (lanes 2, 3, 5, and 6) in the absence (lanes 2 and 5) or presence (lanes 3 and 6) of 1 ug/ml heparin, followed by immunoprecipitation with anti-Myc antibody. Immunopreciptates were analyzed by SDS-PAGE and autoradiography. C, binding competition: 5 ng of 125I-PlGF-2 were incubated in solution without (lane 1) or with 300 ng of pure Myc-tagged NRP1 b1b2 domain (lanes 2–5), in the absence (lanes 1 and 2) or presence (lanes 3–5) of increased concentrations of unlabeled VEGF165 from 5 (1) to 500 ng (100). Incubation was in the presence of 1 ug/ml heparin and was followed by immunoprecipitation with anti-Myc antibody. Immunoprecipitates were analyzed by SDS-PAGE and autoradiography.

Mamluk, R., Gechtman, Z. E., Kutcher, M. E., Gasiunas, N., Gallagher, J., & Klagsbrun, M. (2002). Neuropilin-1 binds vascular endothelial growth factor 165, placenta growth factor-2 and heparin via its b1b2 domain. Journal of Biological Chemistry.