Extended Data Figure 7. PlxnD1 co-localises and associates with members of the junctional mechanosensory complex, and its levels are not regulated by flow, unlike Sema3E.

(a) The descending thoracic aorta or the inner curvature of aortic arches were isolated and prepared en face from wild-type mice and stained for PlxnD1, PECAM-1 and DAPI. Quantification of PlxnD1 levels was performed by fluorescence intensity measurement on ImageJ; 4-6 images were taken on tissue collected from n=4 animals. The data represent mean±SEM. Scale bar represents 20 μm (b) The descending thoracic aorta was isolated and prepared en face from PlxnD1^iECKO mice and stained for PlxnD1 to assess the specificity of the PlxnD1 immunostain, n=3 animals all showing similar result. (c) The descending thoracic aorta or the inner curvature of aortic arches were isolated and prepared en face from wild-type mice and stained for Sema3E, PECAM-1 and DAPI. Quantification of Sema3E levels was performed by fluorescence intensity measurement on ImageJ; 4-6 images were taken on tissue collected from n=3 animals. The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism *p<0.05; scale bar represents 20 μm (d) Mouse ECs were exposed to shear stress for the indicated times or left as static controls before immunoprecipitating PlxnD1 and examining its association with the junctional mechanosensory complex (PECAM, VE-cadherin and VEGFR2) as well as PI3K/p85, n=3