Figure 1.

Defining the C99 minimum boundary for γ-secretase cleavage. (A) An overall view of the γ-secretase Epsilon-Cleavage assay. Once C99-T4L-rTA hybrid protein is cleaved by endogenous γ-secretase, Aβ peptides are released into the culture medium, and the AICD-T4L-rTA hybrid protein is translocated into the nucleus, where it binds tetO and activates luc transcription to generate a luciferase signal as measurement of total C99 cleavage. (B) An overall view of the AlphaLISA assay. The biotinylated anti-Aβ antibody binds to Streptavidin-coated donor beads, and AlphaLISA acceptor beads are directly conjugated to the anti-Aβ antibody. The presence of Aβ peptides brings the two beads into close proximity to generate a light emission signal as measurement of secreted Aβ levels. (C and D) Defining the C-termini of the minimum substrate by γ-secretase Epsilon-Cleavage assay (C) and AlphaLISA assay (D). Cartoon illustration of the C-terminally truncated C99 fragments (right side) and their cleavage efficiencies (left side). The numbers in the cartoon illustration indicate the last residue in each construct. The inlet panel of C shows relative protein expression of some critical boundary constructs, with corresponding construct numbers marked on top of each lane. Aβ₅₅, which contains first 55 amino acids of C99, is the shortest substrate among these C99 C-terminal truncations that retained similar activity as wild-type. Error bars=SEM, n=3, P-values (two-tailed Student's t-test versus WT): ^*P<0.05; ^**P<0.01, ^***P<0.001.