Western blot analysis of subcellular fractions of mouse brain. Thirty μg of total protein were loaded in each lane. A: Localization and predicted molecular masses of the indicated polypeptides were determined using the antibodies listed at right (see text). PM, plasma membrane. B: Fractions were probed using the indicated antibodies against PS1 (Calbiochem PC267) and PS2 (Cell Signaling 2192) and to other components of the γ-secretase complex. In the blots shown here, the intensity of both the PS1 and the PS2 signals in MAM was enriched ∼eightfold over that in the ER. In some blots, data represent nonadjacent lanes taken from a single blot; dividing lines indicate where lanes were pasted together.

γ-Secretase activity assays. A: Activity using a fluorescence based energy transfer-based assay, in the absence and presence of Compound E, a γ-secretase inhibitor.37 Serial dilutions of the indicated subcellular fractions from mouse brain were assayed for APP cleavage activity (in arbitrary units/μg protein). Bars = SD; *P < 0.05 in MAM compared with the other fractions n = 3 for all fractions. B: Αctivity using Western blotting to detect APP intracellular domain,40 in the absence and presence of Compound E. The identity of the lower bands in the first and third lanes is unknown, but may be cross-reaction to another APP-like polypeptide (eg, APLP1, APLP2). The specificity of the APP intracellular domain signal was confirmed in PS1/PS2 double-knockout mouse embryonic fibroblasts (not shown).

Immunocytochemistry to detect FACL4 and presenilins in mammalian cells. A: Double staining of human fibroblasts with MT Red and anti-FACL4. FACL4 apparently colocalizes with MT Red, mainly in regions proximal to the nucleus (yellow arrowhead), with a lower degrees of co-localization in more distal mitochondria (red arrowhead). In an enlarged view of the perinuclear region from another merged field (rightmost panel), note discrete regions where the red and green signals (arrowheads) are in apposition and do not overlap. B: Double staining of human fibroblasts with MT Red and anti-PS1. Note the similarity of the colocalization pattern to that seen with FACL4. C: Double-staining of human fibroblasts with anti-FACL4 (red) and anti-PS1 (green). There is significant overlap between the red and green signals, even in the enlarged merged view of the perinuclear region, implying that both proteins are in the same compartment (ie, MAM). D: Double staining of mouse 3T3 cells with MT Red and anti-PS2. Note the similarity of the colocalization pattern to that seen in A and B. E: Double staining of confluent COS-7 cells with MT Red and anti-PS1, photographed in a plane of focus to reveal the localization of PS1 to adherens junctions (adherens junctions; arrowheads). The MT Red staining is fuzzy because almost all mitochondria are below the plane of focus. Note the absence of colocalization of PS1 with MT Red in adherens junctions. Immunostaining of anti-PS1 (Ab P7854) was suppressed in the presence of the peptide epitope used to generate the antibody, confirming its specificity (not shown).