Fig. 1.

Catalytic inhibition of presenillin-dependent γ-secretase causes accumulation of a 15 kDa MUC1 fragment containing the CTD (CTF15). Cytokine-stimulated HES cells were treated for 64 h with the indicated concentrations of the γ-secretase inhibitors, L685,458 or S2188 (panel A). Not shown are treatments with 0, 0.001, 0.01, and 0.1 µM L685,458 performed in the same experiment which were identical to results shown for 0.5 µM L685,458. Cell lysates were analyzed by SDS–PAGE and Western blotting with CT1 antibody as described in “Experimental Procedures Section.” Inhibitor treatment resulted in dose-dependent accumulation of a MUC1 peptide containing the CTD (panel A), migrating at 15 kDa. Migration of the larger CT subunit of the metabolic complex at approximately 20–23 kDa is indicated by a bracket to the right. Accumulation of the CTF15 was time-dependent in the presence of 10 µM L685,458 (panel B). Performed as described in “Experimental Procedures Section,” densitometric analyses of the accumulation of CTF15 relative to other CT forms are presented to the right of panel B. Total CT is defined as the sum of all bands recognized by CT1 within the sample.