2.8 µm of magnetic protein A–coated beads (Dynabeads; Invitrogen) were coated with Ecad-hFc. In brief, 10 µl of the blurry solution was washed three times and resuspended in 200 µl of 0.1 M borate buffer, pH 8.0, before 2 × 30-s sonication. Then, 50 µl of goat anti–human IgG Fc fragment (2.4 mg/ml; Jackson ImmunoResearch Laboratories, Inc.) was added and left to incubate overnight on a wheel at room temperature. Beads are then washed three times and resuspended in 200 µl PBS (Life technologies) before 2 × 30-s sonication. Then 5 µl of recombinant Ecad-Fc were added and left to incubate for 3 h on a rotating wheel at room temperature. Finally, beads were washed and resuspended in 1 ml PBS supplemented with 1% BSA. 50 µl of this Ecad-coated bead solution was added to cells grown on a 22 × 22-mm glass coverslip placed on a 3.5-cm Petri dish for 1 h. After extensive washes to remove unbound beads, the medium was changed for phenol red–free DMEM supplemented with 20 mM Hepes, pH 7.4.
For bead binding assays, Ecad-hFc– or hFc-coated beads were deposited on nontransfected and wt Ecad– or cis-Ecad–transfected cells, left to adhere for 1 h, and gently washed before fixation. Images were taken with a DM6000 microscope equipped with a 10× objective and a micromax CCD camera. The number of bound beads per squared millimeter was then manually scored.
For bead binding assays, Ecad-hFc– or hFc-coated beads were deposited on nontransfected and wt Ecad– or cis-Ecad–transfected cells, left to adhere for 1 h, and gently washed before fixation. Images were taken with a DM6000 microscope equipped with a 10× objective and a micromax CCD camera. The number of bound beads per squared millimeter was then manually scored.