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Fig. 6. Sec8 associates with a fraction of ZO-2. (A) MDCK cells were extracted in 1% Triton X-100 either 0 hours (contact-naive) or 48 hours (polarized) after inducing calcium-dependent cell-cell adhesion. Extracts were subjected to immunoprecipitation with specific antibodies to Sec8, Exo70, ZO-1, ZO-2 or occludin. The presence of Sec8 in precipitated immune complexes was assessed by SDS-PAGE followed by immunoblotting with Sec8 antibodies. (B) MDCK cells were homogenized 48 hours after induction of calcium-dependent cell-cell adhesion and junction-enriched membrane fractions were isolated by isopycnic density gradient centrifugation as described in Fig. 2. Membranes were extracted in 1% Triton X-100 and subjected to immunoprecipitation with antibodies specific for Sec8, occludin, claudin-1 or claudin-2. The presence of each of these proteins and of ZO-2 in precipitated immune complexes was assessed by SDS-PAGE followed by immunoblotting with specific antibodies. (C) Polarized MDCK cultures on polycarbonate filters were treated with 2 µM latrunculin B for 1 hour, then fixed with 2% paraformaldehyde before extraction with buffer containing 1% Triton X-100. Anti-Sec6 monoclonal antibody (9H5) was visualized with FITC-labeled goat anti-mouse antibody and anti-ZO-2 polyclonal antibody was visualized with Texas Red-labeled donkey anti-rabbit antibody.
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