Figure 3. PlxnD1, NRP1 and VEGFR2 mechano-complex functions upstream of known mechanosensory hotspots and is sufficient for responsiveness to shear stress.

(a) Schematic showing signalling at the junctional complex and integrins. (b) Mouse ECs were transfected with Scr or PlxnD1 siRNA, exposed to shear stress for 2min before immunoprecipitating VEGFR2 and examining its phosphorylation and association with the p85 subunit of PI3K and VE-Cadherin, n=3. (c) Mouse ECs were transfected with Scr or PlxnD1 siRNA, exposed to shear stress for 30min before immunoprecipitating integrin α_Vβ₃ and examining its association with Shc, n=3. (d) ECs treated with VEGFR2 kinase inhibitor SU1498, transfected with siRNA to NRP1 or treated with NRP1 blocking antibody, then incubated with anti-PlxnD1-coated beads and subjected to force for 5min before examining phosphorylation of vinculin. (n=3; *p < 0.05) The data represent mean±SEM. P-values were obtained by performing two-tailed Student's t test using Graphpad Prism. (e) Mouse ECs were exposed to shear stress before immunoprecipitating VEGFR2 and examining its phosphorylation and association with PlxnD1, NRP1 and Src; n=3. (f) Mouse ECs were exposed to shear stress before immunoprecipitating NRP1 and examining its association with PlxnD1 and VEGFR2; n=3. (g) Mouse ECs transfected with either Scr or NRP1 siRNA were exposed to shear stress before immunoprecipitating VEGFR2 and examining its association with PlxnD1; n=3. (h) Schematic showing that reconstitution of PlxnD1, VEGFR2 and NRP1 in Cos7 cells confers shear stress sensitivity to these cells. (i) Cos7 cells were left untransfected or transfected with NRP1 and VEGFR2, with or without PlxnD1 before being subjected to shear stress for 2min and VEGFR2 was immunoprecipitated. Shear stress sensitivity was assessed by examining phospho-VEGFR2 levels, complex formation between VEGFR2 and Src and complex formation of PlxnD1, VEGFR2 and NRP1, n=3. All shear stress experiments were at 12 dynes/cm² using a parallel plate system.