Fig. 7. Sec6/8 complex is associated with E-cadherin and nectin-2. Polarized MDCK cells cultured on polycarbonate filters were surface labeled with either Sulfo-NHS-SS-Biotin (A) or Sulfo-NHS-LC-LC-Biotin (B,C) and extracted with 1% Triton X-100 either directly (no x-link or -DSP) or following (x-link or +DSP) chemical cross-linking with the membrane-permeable cross-linker DSP. Non-biotinylated controls (no biotin) were subjected to cross-linking prior to extraction. (A) Extracts were incubated with avidin-agarose, and the presence of Sec8 in avidin precipitates was assayed by SDS-PAGE followed by immunoblotting with anti-Sec8 antibody. (B,C) Extracts were subjected to immunoprecipitation with anti-Sec8 antibodies, and the presence of biotinylated proteins, Sec8, nectin-2, nectin-1 and E-cadherin in precipitated immune complexes was assessed by SDS-PAGE followed by immunoblotting with HRP-avidin or specific antibodies. `+EGTA' cultures were incubated in LCM + 2 mM EGTA for 6 hours prior to biotinylation.