Fig. 2: ENG and NRP1 form VEGF-A-sensitive heteromeric complexes.
From: NRP1 interacts with endoglin and VEGFR2 to modulate VEGF signaling and endothelial cell sprouting
COS7 cells were co-transfected with vectors encoding myc-NRP1 alone or together with HA-ENG or empty vector (control). After 24 h, live cells were subjected to the IgG crosslinking (CL) protocol (see Methods), resulting in HA-ENG patched and labeled by Alexa 488-GαR IgG (designated CL: IgG αHA), and myc-NRP1 labeled exclusively by monovalent Fab’ (with Alexa 546-GαM Fab’ secondary antibody). In control experiments without crosslinking, HA-ENG was labeled by Fab’ instead of IgGs. Where indicated, ligand (5 ng/ml BMP9, or 50 ng/ml VEGF-A) was added at the last fluorescent labeling step for the FRAP experiment, and maintained at later steps. FRAP studies were conducted as in Fig. 1. Representative FRAP curves of myc-NRP1 coexpressed with uncrosslinked (Fab’-labeled) HA-ENG (a), after IgG-mediated CL HA-ENG (b), and of HA-ENG immobilized by IgG CL (c). Typical curves for the singly-expressed, Fab’-labeled receptors are given in Fig. 1. Average Rf (d) and D values (e) of the effect of HA-ENG coexpression and IgG-mediated immobilization on the lateral diffusion of myc-NRP1. Bars, mean ± SEM. The number of measurements (each conducted on a different cell) is shown under each bar. Some of these numbers are lower in the D panels, since only Rf can be extracted from FRAP curves yielding less than 20% recovery. Asterisks indicate significant differences between the Rf values of the pairs indicated by brackets (*p < 0.05; **p < 0.01; ****p < 10−4; one-way ANOVA and Bonferroni post-hoc test. ns = not significant). A similar analysis of the D values showed no significant differences.
