Figure 4
ILK inhibition facilitated the suppression of MUC1-C expression through ubiquitin-dependent proteasomal degradation. (a) T315 (upper) or shRNA-mediated ILK knockdown (lower) decreased the protein stability of MUC1-C in AsPC-1 and SW1900 cells. Cells were co-treated with T315 (2 μM) and cycloheximide (100 μg/ml) or ILK-depleted cells were treated with cycloheximide (100 μg/ml) for the indicated time intervals. (b) The proteasome inhibitor MG-132 protects AsPC-1 and SW1990 cells from T315-mediated MUC1-C degradation. Cells were treated with 1 μM MG-132 and 2 μM T315, individually or in combination, versus vehicle control for 24 h. (c, d) ILK inhibition via T315 treatment or shRNA-mediated ILK depletion facilitates MUC1-C ubiquitination in AsPC-1 and/or SW1990 cells. Cells ectopically expressing HA-ubiquitin (HA-Ub) were treated with (c) 2 μM T315 for 18 h followed by co-treatment with 1 μM MG-132 for an additional 6 h or (d) 1 μM MG-132 for 6 h. Equal amounts of cell protein lysates were immunoprecipitated with MUC1-C antibody and protein A/G agarose beads followed by immunoblotting with Ub and MUC1-C antibodies. Data shown are representative of at least three independent experiments.
