Ang-1 induces the association of PKCζ with β-catenin in endothelial cells. (A) Immunoprecipitation (IP) of PKCζ from BAEC lysates stimulated with Ang-1 or VEGF and immunoblotted (IB) using anti–β-catenin and anti-PKCζ antibodies. The precipitating antibodies were IgG and anti-PKCζ. Whole-cell lysates were probed for phosphorylated (p-PKCζ) and total PKCζ levels (bottom). (B) Representative confocal micrographs of immunofluorescence staining of BAECs stimulated or not with Ang-1 (30 minutes) using antibodies against endogenous PKCζ (red), β-catenin (blue), and VE-cadherin (green). The overlap (merge) of the 3 fluorophores is shown as white. White arrowheads point to staining at cell-cell junctions. Scale bar represents 20 μm. (C) Representative fluorescence intensity profiles of β-catenin (blue), VE-cadherin (green) and PKCζ (red) measured along the white line in panel B drawn across the cell-cell contact in control and Ang-1–stimulated conditions. Dashed lines delimit the β-catenin staining peak intensity. (D) Representative confocal micrographs of immunofluorescence staining of p-PKCζ (green) and β-catenin (red) in confluent BAECs stimulated with Ang-1 compared with control nonstimulated cells. White arrowheads point to staining at cell-cell junctions. Scale bar represents 20 μm. (E) Representative fluorescence intensity profiles of β-catenin (red) and p-PKCζ (green) measured along the line in panel D drawn across the cell-cell contact in control and Ang-1–stimulated conditions. Dashed lines delimit the β-catenin staining peak intensity. (F) Immunoprecipitation of Flag-tagged WT or DN PKCζ from lysates of transfected BAECs stimulated or not with Ang-1. The coimmunoprecipitated β-catenin was detected by Western blot analysis using anti–β-catenin antibody (top). p-PKCζ was detected in the anti-Flag immunoprecipitates from WT-Flag-PKCζ but not in DN-Flag-PKCζ–transfected cells in Ang-1–stimulated cells (middle). Anti-Flag and anti–β-catenin immunoblots showing equal expression and immunoprecipitation levels are shown (bottom). BAEC lysates (10% input) were immunoblotted with anti–β-catenin antibody. Representative immunoblots from 3 experiments. (G) Immunoprecipitation of Flag-tagged WT, DN, or CA PKCζ from transfected COS-7 cell lysates. Cells were cotransfected with Myc-tagged β-catenin constructs in absence or in presence of the Tie2 expression vector. Anti-Flag immunoprecipitates were immunoblotted with anti-Myc and anti-Flag antibodies to detect β-catenin and PKCζ levels (top and bottom, respectively). Whole-cell lysates were immunoblotted with anti-Myc and anti-Tie2 antibodies for input (10%). Representative immunoblots of 5 experiments. (H) Immunoprecipitation of PKCζ from sparse or confluent BAECs stimulated with Ang-1 or VEGF (left) or after pretreatment with EGTA (30 minutes) to disrupt adherent junctions in confluent cells (right). PKCζ immunoprecipitates were probed for β-catenin and p-PKCζ. Total PKCζ was immunoblotted to show equal immunoprecipitation levels. Representative immunoblots from 3 independent experiments.