Figure 3. Notch1 assembles a mechanosensory junctional complex involving LAR, Trio, and Rac.

a, Fluorescent micrographs of phalloidin stained ECs. b, Intensity of cortical actin at cell-cell junctions and the number of stress fibers per micron quantified from phalloidin stained ECs (n=3 hEMVs). c, Active Rac1 was isolated from scramble and Notch1-KO cell lysates using a recombinant p21-binding domain of Pak1 (GST-PBD). d, Quantification of western blot band intensity revealed a decrease in Rac1 (~50%) after knockout of Notch1 (n=3 independent lysates). e, Active Rac1 was isolated using GST-PBD from Notch1-KO cell lysates expressing mApple or TMD-mApple. f, Quantification of western blot band intensity revealed an increase in Rac1 (~45%) with expression of TMD-mApple (n=3 independent lysates). g, P_D of Notch1-KO ECs expressing TMD-mApple or mApple control in the presence of 50 µM NSC 23766, a Rac1 inhibitor, or vehicle control. h, Immunoprecipitation of VE-cadherin and the Rac1 GEF Trio from ECs cultured statically or under flow. Co-immunoprecipitation of mechanosensory complex proteins was assessed by immunoblotting for Notch1-ECD, Notch1-ICD, LAR (85 kDa P-subunit), and VE-cadherin. i, Immunoprecipitation of VE-cadherin or Trio from scramble and Notch1-KO ECS cultured under flow. Co-immunoprecipitation of mechanosensory complex constituents was assessed by immunoblotting for VE-cadherin, LAR, and Trio. j, P_D of LAR-KO and Trio-KO ECs cultured under static or flow conditions or in the presence of rDll4-coated collagen. k, Micrographs of Trio-KO and LAR-KO cells under flow conditions (VE-cadherin – magenta, actin – green, DAPI – blue). l, Quantification of junctional area measured from VE-cadherin immunostained micrographs. m, Immunoprecipitation of VE-cadherin and Trio from Notch1-KO cells expressing TMD-mApple or mApple. Immunoblotting with LAR, VE-cadherin, and RFP antibodies was used to assess co-immunoprecipitation of mechanosensory complex upon expression of TMD. n. P_D for scramble, N1KO, LAR-KO, and Trio-KO cells cultured under static conditions expressing TMD-mApple or mApple infection control. For (g,j,n), n≥3 hEMVs. All plots mean ± s.e.m., *p<0.05, **p<0.01. Exact p and n values available in Figure Source Data, all images representative of at least three independent experiments.