Fig. 6.

Proteasomal inhibition causes accumulation of MUC1 CTF15. In panel A, HES cells were exposed to cytokines plus the reversible 7–secretase inhibitor, S2188 (10µM), for 56 h. S2188 then was removed and culture continued for the indicated times in the presence of cytokines and the absence (−) or presence (+) of the proteosomal inhibitor, MG132 (10µM), for the indicated times. CTF15 was detected by antibody CT1. Triplicate independent samples were analyzed in each case. Note that while CTF15 is lost upon removal of the S2188 it continues to accumulate in the presence of MG132. Panel B unstimulated or cytokine-stimulated HES cells were cultured with or without 10µM MG132 for 14 or 26 h. CT forms were detected in cell lysates by antibody CT1. Densitometric analysis demonstrates that within the treatment period used in panel A experiments, proteasomal inhibition by MG132 effectively blocked cytokine induction of all forms of CT (Total CT) and was capable of progressive reduction of constitutive expression. Note that CTF17 and CTF15 are undetectable in unstimulated HES cells. Densitometric analysis of CT forms expressed in response to cytokine stimulation reflect the relative concentrations of CT forms that would be produced during the same time frames.