Fig. 2. Sec6 co-localizes with components of the nectin complex in early cell-cell contacts and polarized MDCK cells. (Top panels) Low-density cultures of MDCK cells were allowed to form calcium-dependent cell-cell contacts for 1 hour, and then fixed with 2% paraformaldehyde before extraction with 1% Triton X-100. Sec6 distribution was compared to that of afadin or nectin-2. (Bottom panel) Confluent MDCK cultures on polycarbonate filters were fixed and extracted 24 hours after induction of calcium-dependent cell-cell adhesion. Sec6 distribution was compared to that of afadin. Anti-Sec6 monoclonal antibody (9H5) was visualized with FITC-labeled goat anti-mouse antibody (in the afadin panels) or with Texas Red-labeled donkey anti-mouse antibody (in the nectin panel). Rabbit polyclonal antibody to afadin was visualized with Texas Red-labeled donkey anti-rabbit antibody, and rat monoclonal antibody to nectin-2 was visualized with FITC-labeled goat anti-rat antibody. Confocal images were obtained as described in Fig. 1 legend. Scale bar: 10 µm.