Pulse-chase experiments were performed exactly as described previously (Ireton et al., 2002). Biotinylation and the rate of cell surface trafficking were also performed exactly as described previously (Bonifacino et al., 2003). In brief, cells were plated at 5 × 105 cells per 60-mm dish for 36 h before pulse chase. Cells were 35S-labeled for 15 min before chase. At the end of the chase, cell surface proteins were labeled with 1 mg/ml EZ-Link sulfo-NHS-SS-biotin (Pierce Chemical Co.) at 4°C for 30 min. E-cadherin was immunoprecipitated from NP-40 cell lysates, and surface cadherin was detected by dividing the E-cadherin immunoprecipitations in half, eluting E-cadherin from the beads with 0.5% SDS, reconstituting elutions in 50 mM Tris-HCl (pH 7.5), and pulling down biotinylated E-cadherin with 10 μl per sample of packed streptavidin-coated agarose beads (Sigma-Aldrich) for 1 h at 4°C. Samples were washed three times with 50 mM Tris-HCl (pH 7.5), and protein was eluted with 2× Laemmli sample buffer and analyzed by SDS-PAGE and autoradiography as described previously (Ireton et al., 2002). Quantification was performed by densitometry using Image Gage software
