To definitively address this issue, we combined the pulse-chase and biotin surface-labeling strategies in order to selectively examine the fate of the nascent E-cadherin molecules with respect to their arrival at the cell surface (Fig. 5 c). The pulse labeling was conducted as in Fig. 5 a, except that surface E-cadherin was subsequently biotin labeled (as in Fig. 5 b) at each time point after the pulse chase. The surface-labeled cadherins were then isolated by streptavidin pulldown, and nascent cadherins were visualized by SDS-PAGE and autoradiography. The data show that the rate of nascent E-cadherin arrival at the cell surface is almost identical in the presence and absence of p120 (Fig. 5, c and d; compare parental and siRNA cell lines). The appearance and removal of E-cadherin from the cell surface (Fig. 5 c; E-cadherin + streptavidin immunoprecipitations) are quantified by densitometry and displayed graphically in Fig. 5 d. Note that peak levels of nascent (35S-labeled) E-cadherin at the cell surface occurred at 1 h, and by 4 h, the nascent cadherin was either moving off the surface or getting
