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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.
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