Fig. 6. Overexpression of Dsh causes a reduction in the stability of newly synthesized E-cadherin. cl8mEcad cells and cl8HSDsh/mEcad cells induced to overexpress Dsh were metabolically labeled and cell lysates were immunoprecipitated for E-cadherin, separated by SDS-PAGE and processed for fluorography. (A) Both cell lines exhibited comparable amounts of newly synthesized E-cadherin after 2 hours of labeling with [³⁵S]methionine (0 hours chase, lanes 1 and 6). Note that E-cadherin is synthesized as a 135 kDa precursor polypeptide, which is later proteolytically processed to generate the mature 120 kDa protein (Shore and Nelson, 1991). Whereas conversion of precursor to mature E-cadherin was clearly visible in cl8mEcad cells during the chase period (lanes 2-5), very little mature E-cadherin was detectable in cl8HSDsh/mEcad cells (lanes 7-10). The half-life of E-cadherin in cl8HSDsh/mEcad cells was much shorter than in cl8mEcad cells, indicating a rapid turnover of E-cadherin as a consequence of Dsh overexpression. Note that Arm and -catenin were not detectable in this experiment, presumably because the relative amount of newly synthesized E-cadherin was much higher than that of Arm and -catenin during the labeling period. (B) Relative intensities of the slower migrating 135 kDa precursor band (squares) and the faster migrating band representing 120 kDa mature E-cadherin (triangles). Intensities of both bands were measured by densitometry and plotted as the ratio between the measured intensity and the intensity of the precursor band at time 0. In cl8mEcad cells, the intensity of the mature 120 kDa band increased rapidly between 0 and 1 hour chase and had decreased only by 30% after 4 hours of chase, whereas in heat shocked cl8HSDsh/mEcad cells, very little mature protein was detected.