Expression of PS1 and PS2 in WT and asp-mutant PS1/PS2 cells. (a) CHO cells stably expressing WT PS1, D385A PS1, or D257A PS1 plus D366A PS2 were lysed and immunoprecipitated (IP) with PS1 polyclonal antibodies X81 to the NTF, R22 to both NTF and CTF, or preimmune serum (pre) from rabbit X81. The precipitates were probed by Western blotting (WB) with PS1 mAb 13A11 to the CTF. (b) Lysates from untransfected cells or PS1/PS2 double asp-mutant cells were immunoprecipitated and Western blotted with PS2 antibody 2972.

Enrichment of PS/APP-CTF complexes in Golgi/TGN-rich subcellular fractions. (a) Distribution of the ER marker protein calnexin in discontinuous Iodixanol gradient fractions prepared from cells stably expressing WT PS1 was detected by Western blotting (WB) with anticalnexin antibody (densest fraction, lane 1; lightest fraction, lane 12). (b) Distribution of the Golgi/TGN marker β-1,4-galactosyltransferase activity was mainly in fractions 4–8 of WT PS1 cells. Curve represents the best fit for mean levels of four determinations (symbols). (c) Individual subcellular fractions (1–12) from cells expressing WT human PS1 were coimmunoprecipitated with PS1 antibodies X81 + 4627 (Right) or preimmune serum (Left), followed by Western blotting with APP antibody 13G8. FL N-APP that coimmunoprecipitated with PS1 is shown in the Upper Right (fractions 1–4); coprecipitating C83 is shown in the Lower Right (fractions 5–8). These coprecipitates comigrate, respectively, with the FL-APP and C83 species immunoprecipitated with APP antibody C7 (far right lane). (d) Individual subcellular fractions (only fractions 4–8 are shown) from the asp-mutant 3–1 cells (Left) or 2A-2 cells (Right) were coimmunoprecipitated with PS1 antibodies (X81 + 4627) or PS2 antibodies (2972 + PS2L), respectively. Fraction 6 was divided into two equal aliquots that were precipitated either with the indicated immune sera or with preimmune serum (Pre). APP antibody C7 was used to precipitate C83 and C99 as markers (far right lane). Precipitates were probed by Western blotting with APP antibody 13G8. (e) Individual subcellular fractions from human cells expressing endogenous levels of PS (cell line 293_695SW) were coimmunoprecipitated with PS1 antibodies X81 + 4627 followed by Western blotting with 13G8. Fraction 6 was divided into two equal aliquots and precipitated with the immune sera or with a preimmune serum (Pre). FL human N-APP₆₉₅ that coprecipitated with PS1 is shown (Upper) (fractions 1–3; the band below APP₆₉₅ is nonspecific); the coprecipitating C83 is shown (Lower) (fractions 5–7).

Accumulation of PS/APP-CTF complexes after inactivation of γ-secretase in stably transfected cells. (a) Lysates from PS WT (lanes 5–8) and asp-mutant (lanes 1–4) stable transfectants were coimmunoprecipitated (IP) with either PS1 antibodies (X81, 4627, or R22) or preimmune serum (pre), as indicated, followed by Western blotting (WB) with APP antibody 13G8. The APP species that coprecipitated with both WT and asp-mutant PS1 comigrated with the N-glycosylated form of APP detected on straight Western blots of the lysates (lanes 9–11). The lower band in lanes 2–4 and 6–8 is nonspecific. (b) Cell lysates were coimmunoprecipitated with PS1 antibodies (combined X81 and 4627, lanes 1, 3, and 5), PS2 antibody 2972 (lane 7), or preimmune serum (lanes 2, 4, 6, and 8). Precipitates were probed by Western blotting with APP antibody 13G8. The C83 and C99 that coprecipitate with PS1 and PS2 are only a small fraction of the C99 and C83 detected by straight Western blotting (WB) of 1/20th the amount of the respective cell lysates with 13G8 (lanes 9–11). (c) Structure of the difluoro ketone peptidomimetic γ-secretase inhibitors, compounds 1 and 11. (d) Lysates from WT PS1 cells treated with vehicle alone (DMSO) or the reported γ-secretase inhibitor Cpd 11 for 8 hr were coimmunoprecipitated with PS1 antibody R22 (lanes 3 and 5) or preimmune serum (lanes 2 and 4). Precipitates were probed by Western blotting (WB) with APP antibody C7 to detect C83. Small amounts of C99 (which is substantially less abundant than C83) also can be coprecipitated with PS1, but this is very difficult to visualize photographically. Lane 1 shows a straight C7 Western blot of a D257A PS1 cell lysate to indicate the gel positions of C83 and C99.

Steady-state Aβ levels and de novo Aβ generation in isolated subcellular fractions (a) WT and the indicated asp-mutant CHO cells were subjected to subcellular fractionation, and steady-state Aβ levels in each fraction were measured by sandwich ELISA (n = 3 or 4). (b) To measure de novo Aβ generation, each individual fraction was collected and incubated at either 37°C or −80°C (base line) for 4 hr. Levels of newly generated Aβ were established by subtracting the Aβ level at −80°C from that generated at 37°C (n = 8–10). Standard errors are indicated on each bar.