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. 2000 Aug 1;97(16):9299–9304. doi: 10.1073/pnas.97.16.9299

Figure 3.

Figure 3

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 293695SW) 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).