Fig. 1

Sec6/8 complex becomes restricted to apical junctional complex during development of cell polarity. Contact-naive MDCK cells were seeded at confluent density on collagen-coated filters, allowed to attach in low calcium medium for 3 hours, and then switched to high calcium medium for 0, 1, 3, 6, 12, or 24 hours. At each time point, cultures were fixed with 4% paraformaldehyde and extracted with buffer containing 1% Triton X-100. (A,B) Sec6 distribution was compared to that of either E-cadherin (A) or ZO-1 (B). Anti-Sec6 monoclonal antibody (9H5) was visualized with FITC-labeled goat anti-mouse antibody. Rabbit polyclonal antibodies to E-cadherin and ZO-1 were visualized with Texas Red-labeled donkey anti-rabbit antibody. Confocal images in the upper panels were acquired along the x–y axis (en face view) of the cell monolayer. The x–z views, in the lower panels, were constructed by averaging sections over a line at each z position in 0.2 μm steps. Scale bar: 10 μm. (C) Relative pixel intensities of Sec6, E-cadherin and ZO-1 fluorescence at each optical section (1=apical, 19=basal) were averaged from five independently scanned fields.