Figure 1.

C50 is more analogous to endogenous AICD than C60. a, Comparison of size and Fe65-mediated stabilization of C60 and C50. Lanes 1 and 2 are consistent with previous experiments showing that coexpression of C60 with Fe65 results in additional stabilization (Cupers et al., 2001; Kimberly et al., 2001). C50 is similarly stabilized by Fe65, but its steady-state level is substantially lower (lanes 3, 4). Identical COS7 cultures were transiently transfected with the indicated constructs. Five times the amount of protein was loaded in lanes 3 and 4 than in lanes 1 and 2, allowing detection in the latter lanes of the endogenous COS APP CTFs (bracket) and some nonspecific bands, all of which were also visible during overexposure of lanes 1 and 2 (data not shown). b, The effect of protease inhibitors on C50. COS7 cells were transfected with the indicated constructs and lysed in 1% SDS lysis buffer containing complete protease inhibitors (Roche) and the indicated additional inhibitor or solvent. The proteasome inhibitor mixture (protea.) contained 10 μm MG132, 10 μm lactacystin, and 10 μm LLnL. 1,10-Phenanthroline (1,10-ph) was shown previously to stabilize AICD in an in vitro membrane reaction (Edbauer et al., 2002). Longer exposures revealed faint C50 bands in lanes 1 and 2. c, C50, but not C60, comigrates with endogenous AICD from a stable CHO cell line overexpressing wild-type APP₇₅₁ (7wB4). Fe65 stabilizes the endogenous AICD (lane 2), which comigrates with C50. V, Vector. d, Endogenous AICD formation is prevented by a γ-secretase inhibitor. 7wB4 cells were transfected with Fe65 and then treated with the γ-secretase inhibitor DAPT (or DMSO) for 4 h.