Effect of Z-IE(Ot-Bu)A-Leucinal on Aβ40 and Aβ42 secretion by HEK293 cells expressing wild-type and FAD-linked PS1. Naive HEK293 cells or stable transfectants overexpressing wt or the indicated mutated PS1 proteins were transiently transfected with 2 µg of wild-type βAPP751 cDNA. Thirty-six hours after transfection, cells were incubated for 16 hr with Z-IE(Ot-Bu)A-Leucinal (25 µM), then metabolically labeled for 6 hr in the presence of the inhibitor. Conditioned media were submitted to a subsequent two-step immunoprecipitation with FCA3542 (A) and FCA3340 (B) antibodies (350-fold dilution) as described in Materials and Methods. Immunoprecipitated proteins were electrophoresed on a 16.5% Tris-tricine gel and radioautographed and analysed by densitometry. Panel D represents the ratio of Aβ42 to total Aβ (corresponding to Aβ42 + Aβ40) observed with indicated clones in the absence (control, black bars) or presence (Z-IE(Ot-Bu)A-Leucinal, white bars) of the proteasome inhibitor. Values are the means ± S.E.M. of three independent experiments. Panel C represents βAPP immunoreactivity revealed with mAb22C11 as described in Materials and Methods.