FIGURE 4:

Production of the 90-kDa E-cadherin fragment depends on proteasome and Hakai. (A) Effects of proteasome inhibitors on 90-kDa fragment production. Caco-2 cells were transfected with control or KIFC3-specific siRNA. At 7 h after transfection, cells were passaged. After 24 h, dimethyl sulfoxide, 1 μM MG132, 10 μM lactacystin, or 100 nM epoxomycin was added to culture medium. At 16 h later, cells were harvested and subjected to Western blotting assay using the antibodies indicated. (B) Effects of Hakai depletion on 90-kDa fragment production. At 48 h after transfection of cells with indicated siRNAs, cells were harvested and subjected to Western blotting assay using the antibodies indicated. Knockdown efficiency for each molecule is shown on the far right. (C) Effects of proteasome and lysosome inhibitors on 90-kDa fragment production. Cells were treated with siRNAs and subsequently with 10 mM NH₄Cl and 21 μM leupeptin with or without 100 nM epoxomycin, according to the same protocol as in A.