Ang-1 induces the colocalization of polarity and adherens junction proteins at the leading edge of migrating endothelial cells. (A) Representative immunofluorescence micrographs showing colocalization (merge; right) of β-catenin, VE-cadherin, and PKCζ at the leading edge of migrating BAEC subjected to Ang-1 stimulation (30 minutes). Scale bar represents 20 μm. Higher magnification views of the boxed region are shown. (B) Quantification of the overlap coefficient of colocalization for PKCζ/β-catenin, PKCζ/VE-cadherin or β-catenin/VE-cadherin in control (white) and Ang-1–stimulated cells (black) at the leading edge (LE) and at cell junctions (CJ). (C) Representative immunofluorescence microscopy images of BAECs stained using anti-PKCζ showing increased colocalization for PKCζ, Par3, and Par6 in Ang-1–stimulated cells (30 minutes). Higher magnification view of the boxed region is shown. Scale bar represents 20 μm. (D) Quantification of the overlap coefficient of colocalization for PKCζ/Par6, PKCζ/Par3 in control (white) and Ang-1–stimulated cells (black) at the LE and at CJ. Each column represents the average of at least 36 measurements, and data are represented as mean ± SEM (*P < .05 compared with nonstimulated cells). (E) Immunoprecipitation of endogenous PKCζ from BAECs stimulated or not with Ang-1. PKCζ immunoprecipitates and total cell lysates were subjected to Western blot analysis with anti–β-catenin, anti–VE-cadherin, anti–α-catenin, anti-Par6, anti-Par3, and anti-PKCζ antibodies. Ratios of the densitometric analyses of immunoblots are presented. (F) Immunoprecipitation of PKCz from BAECs, as in panel E, transfected with CT or β-catenin-siRNA.