Fig. 5: Identification of the LRG1-LPHN2 signaling pathway.

a HUVECs were stimulated with LRG1 (1 μg/ml) for the indicated times under high-glucose conditions. Immunoprecipitants with an anti-LPHN2 antibody were analyzed by Western blotting using antibodies specific for pY-LPHN2 and LPHN2. b Cignal Finder GPCR signaling 10-pathway reporter array analysis of HUVECs after treatment with LRG1 (1 μg/ml). The results of dual-luciferase assays are presented as normalized relative luminescence signals (means ± SEM, n = 3). The relative ratio of the untreated group was defined as 1. **P < 0.01 (Student’s t test). c Network model showing interactions between the proteins with increased phosphorylation in response to LRG1. Node color indicates the increase (red) in the phosphorylation level. The color bar indicates the gradient of the log2-fold-change of phosphorylation levels by LRG1 with respect to those in untreated control conditions. Circled P on a node indicates phosphorylation of the corresponding protein. Arrows, activation; inhibition symbols, inhibition; solid arrows, direct activation; dotted arrows, indirect activation; gray lines, protein–protein interactions; and green lines, plasma membrane. d Western blot analysis of HUVECs stimulated with LRG1 (5 μg/ml) using the indicated antibodies. e Western blot analysis of HUVECs stimulated with 1 µg/ml LRG1 with or without pre-treatment with PP2 (10 μM) or LY294002 (10 μM). f and g HUVECs (f) and mouse DRG explants (g) were treated with LRG1 (1 μg/ml), LY (10 μM), PP2 (10 μM), LRG1 (1 μg/ml) + LY (10 μM), or LRG1 (1 μg/ml) + PP2 (10 μM). Top: Representative images of HUVEC tube formation (f) and βIII-tubulin staining in mouse DRG explants (g). The relative ratio of the untreated group was defined as 1. Scale bars, 100 µm. Bottom: Master junctions (n = 4) (f) and βIII-tubulin–immunopositive axon lengths in DRGs (g) were quantified using ImageJ, and the results are presented as the means ± SEM (n = 4). h Western blot analysis of HUVECs stimulated with 1 µg/ml LRG1 for 30 min or 7 hr using antibodies specific for angiogenic factors (VEGFA, angiopoietin-1, FGF2).