Effects of point mutations
in APP and CTF on the production of
Aβ and AICD, respectively. (A) The WT APP and mutant APP sequences
examined in this study are highlighted. The 3xE construct has been
tested only in the in vitro assay (marked with an
asterisk). (Attempts to produce a stable cell line of the K624E/G625E/A626E
construct were not successful.) (B) The WT APP and mutant APP were
stably expressed in CHO cells and detected via Western blotting using
a 6E10 monoclonal antibody.74 (C) Aβ
spectra obtained by MALDI-TOF analysis of conditioned media from CHO
cells overexpressing WT and mutant forms of APP. Aβ isoforms
are identified on the profiles with nonspecific peaks denoted with
an asterisk. (D) Stacked bar graphs indicating the percent of each
Aβ isoform derived from WT and mutant APP. These analyses were
based on two to four experiments with two to five replicates in each
experiment (the maximal SEM = ±2.5). (E) AICD spectra of WT CTFβ
tagged with a Flag peptide (C100Flag), 3xK CTFβ tagged with
Flag (3xK-C100Flag), and K28E CTFβ tagged with Flag (K28E-C100Flag)
after immunoprecipitation and MALDI-TOF analysis. The positions of
the two major products produced by γ-secretase cleavage at the
ε-site, AICD50–99 and AICD49–99, are indicated.
GSI treatment served as a control to select specific AICD peaks (data
not shown).