Supplementary Figure Legends
Identification of a mechanosensory complex that mediates the endothelial cell response to fluid shear stress. Eleni Tzima et al
Supplementary Figure 1 Activation of integrin avb3, PI 3-kinase and Src by shear stress
(a) Activation of PI 3-kinase after shear stress. The p85 subunit of PI 3-kinase was immunoprecipitated and analyzed by Western blotting with antibodies to phosphotyrosine (4G10) or p85. (b) Confluent monolayers transiently transfected with the GFP-PH domain from Akt. Membrane translocation of the GFP-PH domain (arrows) indicates accumulation of PI 3-lipids. (c) Conversion of avb3 to its high affinity conformation is dependent on PI 3-kinase. BAECs were pretreated with or without LY294002, shear stress applied for the indicated times, then incubated with WOW-1 to label activated avb3. Values are means ± SEM, n=4. (d) Activation of Src was assayed by Western blotting for phosphorylated Tyr418, which increases, and Tyr527, which decreases during activation. Values are means ± SEM, n=4. (e) Shear stress activation of avb3 and PI 3-kinase is Src-dependent. BAECs were treated with PP2 prior to application of shear stress. WOW-1 binding and phosphorylation of p85 were assessed as before. Activation of Src was assayed by Western blotting for phosphorylated Tyr418. Values are means ± SEM, n=4.
Supplementary Figure 2 Recruitment of actin by beads
BAECs were plated at low density. Cells were incubated for 30 min with magnetic beads coated with non-specific mouse IgG or antibodies to PECAM-1 or VE-cadherin. Cells were fixed and stained with TRITC-phalloidin. Insets show phase and fluorescence images of areas within the square at higher magnification. Fluorescence around the beads was scored (100 cells per condition per experiment, n=2).
Supplementary Figure 3 WOW-1 localises near cell-cell junctions
BAECs without or after 1 min flow were fixed and stained with WOW-1 Fab. Arrows indicate higher WOW-1 staining at cell-cell junctions.
Supplementary Figure 4 Role of VEGFR2 in shear stress signalling
(a) BAECs or knockout ECs were sheared, fixed and stained for b-catenin and phospho-Tyr1054 VEGFR2. (b) BAECs with or without pretreatment with the VEGFR2 tyrosine kinase inhibitor VTI were lysed, PI 3-kinase immunoprecipitated and analyzed for tyrosine phosphorylation or p85 by Western blotting. Values are means ± SEM, n=3 **P <0.01.
Supplementary Figure 5 Transfection of Cos7 cells
Cos7 cells were untransfected or transiently transfected with PECAM-1, VE-cadherin and VEGFR2, together with GFP as a marker for transfection. (a) Cells were fixed and stained for PECAM-1, VE-cadherin or VEGFR2. Approximately 80% of the GFP-positive cells were positive for each receptor. (b) Cells were lysed and analysed for expression of the receptors by Western blotting. Protein loading was assessed by immunoblotting for tubulin.
Supplementary Figure 6 Integrin activation requires b-catenin
b-catenin null (KO) and reconstituted (RC) ECs were subjected to shear stress for the indicated times and incubated with WOW-1 to label activated avb3. Values are means ± SEM, n=2.