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. Author manuscript; available in PMC: 2005 Sep 13.
Published in final edited form as: J Biol Chem. 2005 May 10;280(27):25892–25900. doi: 10.1074/jbc.M503570200

Fig. 4. APP CTFs associate with DIM in cholesterol-sensitive manner and co-reside with syntaxin 6 and PS1.

Fig. 4

A, N2a Swe.10 cells were treated with Me2SO or 1 μM L685,458 for 16 h. Lipid raft association of full-length APP and APP CTFs was assessed by flotation sucrose density gradient centrifugation. Note the marked accumulation of APP CTFs in raft fractions in cells treated with the inhibitor. B, Swe.10 cells were exposed to 10 nM CompE for 16 h and then treated with Me2SO (vehicle control) or 5 mM MβCD for 2 h at 37 °C prior to fractionation. Note that DIM accumulation of APP CTFs is sensitive to cholesterol depletion. C, pooled fractions 4 and 5 from the indicated cell lines were fractionated on 16.5% Tris-Tricine gels and sequentially probed with APP C-terminal polyclonal antibody 369 and mAb 26D6 (raised against epitopes 1–12). Antibody 369 reacts with β-, β′ (+11)-, and α-CTFs, whereas mAb 26D6 only reacts with 3-CTF. D, DIM fractions from Swe.10 cells treated with CompE were incubated with magnetic beads coated with syntaxin 6 or OKT8 antibody. Bound DIMs were analyzed by Western blotting using 369, mAb 26D6, and PS1NT. An aliquot of the input (1/30th volume) was also fractionated in the same gel for comparison. Note that APP CTFs, but not full-length APP, co-reside with syntaxin 6 in DIMs.