Co‐IP from HUVECs. Blots for β‐actin shown in (A) were used to confirm equal loading in (B), as the same cell lysates were analyzed in both experiments.
Immunoblots in HUVECs.
Immunoblots in siRNA‐transfected HUVECs.
Co‐IP from siRNA‐transfected HUVECs. The graph shows relative levels of PlexinD1‐VEGFR2 association, in which the values at 0 min are set to 1. n = 3 per group.
Super‐resolution images of receptor colocalization in siRNA‐transfected HUVECs 30 min after Sema3E stimulation. Left panels show immunolabeling of VEGFR2 (green), PlexinD1 (red), and Nrp1 (white), with light‐blue asterisks indicating nuclei. Middle panels show magnified views of white boxes in the left panels. Right panels represent fluorescence intensity profiles along 5‐μm arbitrary lines in the middle panels. Scale bar, 10 μm. The graph shows the proportion of receptor complexes in PlexinD1‐positive vesicles. n = 10 cells per group.
Trajectory plots (left) and rose plots (right) of HUVECs pre‐treated with the p38 MAPK inhibitor SB203580 under Sema3E gradient for 12 h. Red trajectories indicate cells with displacement to the negative y‐axis at the end of analyses. P values for the Rayleigh test represent non‐random distributions of cell endpoints. The graphs show FMI along the y‐ and x‐axes during cell migration. n = 90 cells per group.
A model of Sema3E‐induced reverse cell migration.
Data information: Data represent mean ± SEM (D and F) and mean (E). *
P < 0.05; ***
P < 0.001; NS, not significant, by the Tukey–Kramer test (D) and Student's
t‐test (E and F).
Source data are available online for this figure.
