FIG. 6.

MDM2 associates with and ubiquitinates E-cadherin in the early endosome. (A) MCF-7 cells were treated with MG132 for 4 h, and cell fractionation was performed as described in Materials and Methods. EE, early endosome; LE, late endosome. (B and C) MDM2 associates with E-cadherin (E-cad) in the early endosome. The early endosomes from MCF-7 cells and MDA-MB-468 cells were isolated as described above and then coimmunoprecipitated and immunoblotted with the indicated antibodies. Immunoglobulin G (IgG) was used as a control. (D) Early endosomes from MCF-7 cells were subjected to electron microscopy staining with an anti-MDM2 antibody (larger particle, arrow) and an anti-E-cadherin antibody (smaller particle, arrowhead). (E) HEK 293T cells were transiently transfected with the indicated expression plasmids. The early endosomes were recovered as described above and then subjected to pull-down with Ni-NTA agarose beads followed by SDS-PAGE and immunoblotting with an anti-HA antibody. Filters were stripped and reprobed with an anti-Myc antibody. The relative intensity was calculated by the entire signal from an anti-HA blot divided by the signal from an anti-Myc blot.