Fig. 2. Expression and localization of WAVE2 in epithelial MDCK cells. (A) Comparison of WAVE1 and WAVE2 expression in MDCK cells and mouse embryonic fibroblasts (MEFs). Cell lysates were immunoblotted with anti-WAVE1 and anti-WAVE2 antibodies. (B) Localization of endogenous WAVE2 in MDCK cells. Before (No chelate) or after (Chelate) treatment with EGTA, MDCK cell layers were fixed and then stained with anti-E-cadherin and anti-WAVE2 antibody. The regions from which vertical sections were taken are indicated by white lines. Arrows indicate cell-cell contacts. Bar, 10 µm. (C) Detergent solubility of WAVE2. MDCK cells were treated with 0.5% Triton X-100 containing buffer before fixation. Fixed cell layers were stained with anti-E-cadherin and anti-WAVE2 antibody. Scale bar, 15 µm. (D) Interaction of WAVE2 with adhesion complex proteins. Protein complexes were immunoprecipitated from lysates of DMSO- or cytochalasin D (CytoD)-treated MDCK cells with anti-WAVE2 antisera. Immunoprecipitates were immunoblotted for E-cadherin, ß-catenin, afadin and WAVE2. Negative controls for immunoprecipitations were pre-immune antisera (Pre-Immune IP). (E) DECMA-1 (anti-E-cadherin antibody) inhibits recruitment of WAVE2 to cell-cell contacts. Trypsinized cells were plated on coverslips and treated with DECMA-1. After incubation for 12 hours, cells were fixed and stained for E-cadherin and WAVE2. The lower panels are magnified images of the areas indicated by white squares in each upper panel. Broken lines indicate localization of WAVE2 at the lamellipodial edge. Bars, 15 µm (upper panels); 5 µm (lower panels).