Figure 7: Ang1 induction of β-catenin occupancy at Dll4 promoter and enhancers requires ERG.

ChIP-qPCR analysis of siCtrl and siERG-treated HUVEC, in the presence or absence of Ang1 (250 ng ml⁻¹). Chromatin was immunoprecipitated with (a) an anti-NICD antibody, (b) an anti-β-catenin antibody or control IgG. Immunoprecipitated DNA was analysed by qPCR with primers to −16 kb, −12 kb, promoter, intron 3 and +14 kb of Dll4 locus. Primers covering a negative control region within exon 11 were also used. Results are expressed as fold change enrichment compared to IgG (n=3). (c) HUVEC lysates were immunoprecipitated with an anti-ERG antibody. Immunoprecipitates were analysed by immunoblotting (IB) with anti-ERG, anti-NICD and anti-β-catenin antibodies. (d) Confluent HUVEC were pre-treated with LY294002 (20 μM) or Akt inhibitor IV (8 μM) and treated with Ang1 (250 ng ml⁻¹) or DMSO for 30 min. Proximity ligation assay analysis of localization of ERG-β-catenin interaction was performed using rabbit anti-ERG and mouse anti-β-catenin antibodies (n>99 cells quantified per condition). Scale bar, 20 μm. All graphical data are mean±s.e.m., *P<0.05, **P<0.01, ***P<0.001, Student's t-test.