Fig. 3: LRG1 N325 is the key glycosylation site for attenuating LRG1/LPHN2-mediated angiogenesis and neurite outgrowth.
From: Crystal structure of LRG1 and the functional significance of LRG1 glycan for LPHN2 activation
a Tube formation assay using HUVECs after treatment with 1 μg/ml LRG1, DG-LRG1, or LRG1 glycan mutants (N79D, N186D, N269D, N325D). Representative images of tube formation and Transwell cell migration. Scale bars, 200 µm (top). Bottom, quantification of the number of master junctions and migrated cells using ImageJ. b βIII-tubulin immunostaining in mouse DRG explants after treatment with 1 μg/ml LRG1, DG-LRG1, or LRG1 glycan mutants (N79D, N186D, N269D, N325D). Representative images of DRG explants. Scale bars, 200 µm (top). Bottom, quantification of βIII-tubulin-immunopositive neurite length from DRG explants (200 cells/field) using ImageJ. In the bar graphs, data are plotted as the mean ± SEM (n = 4). Significance is indicated using Student’s t test (***p < 0.001). The relative ratio of the nontreated group was defined as 1. N.S., not significant. Western blot with HUVECs (c) and mouse primary DRG explants d after treatment with LRG1 (1 μg/ml) or LRG1 N325D mutant (1 μg/ml) in the presence of shCon vs. shLPHN2 lentivirus (n = 3).
