Fig. 6

DR6 and TROY Physically and Genetically Interact and Are Required for VEGF-Mediated JNK Activation (A) DR6 physically interacts with TROY. Epitope-tagged receptors were overexpressed in COS-7 cells and flag-tagged TROY bait was immunoprecipitated. Input was 1% total cellular lysate used in pulldown. IP, immunoprecipitation; IB, immunoblot. Results representative of at least three independent experiments are shown. (B) Anti-DR6 function-blocking antibodies, which inhibit human brain endothelial sprouting, also reduce DR6 and TROY physical interaction. Pulldown assays were carried out as described above with the addition of anti-DR6 antibodies four hours after transfection. Relative TROY coimmunoprecipitation was calculated by normalization to both TROY-HA input and DR6-flag pulldown levels. Results representative of at least three independent experiments are shown. (C) Suboptimal concentrations of DR6- and TROY-targeting morpholinos unmask genetic interactions. Each embryo was treated with 4 ng of each splice-blocking morpholino. Endothelial cells are green, DAPI is blue, rhodamine-dextran is red. Results representative of at least three independent experiments are shown. (D) Quantification of DAPI leakage reveals DR6 and TROY genetic interaction. n = 8 (control_MO), n = 8 (DR6_MO), n = 8 (TROY_MO), n = 8 (DR6_MO and TROY_MO). (E) VEGF activates JNK in HBMECs and requires DR6 and TROY expression. Relative protein phosphorylation was calculated by normalization to total protein levels. Results representative of at least three independent experiments are shown. (F) JNK activity is required for HBMEC sprouting. 1 μM JNK inhibitor V or VIII was used. Data are presented as the mean ± SEM (^***p < 0.005; ^**p < 0.01; ^*p < 0.05). See also Figure S6.