Figure 1: Fractionation of β-amyloid-laden APP23 transgenic brain and identification of in vitro seeding-active membrane-associated Aβ.
From: Highly potent intracellular membrane-associated Aβ seeds
(a,b) Supernatant (S) and membrane pellets (P) from brain after differential centrifugation at 1,200 × g (S1,200 × g), 10,000 × g (S10,000 × g and P10,000 × g) and 135,000 × g (S135,000 × g and P135,000 × g) (n = 6; always refers to independent fractionations from different brains) and (c–f) fractions from flotation sucrose density gradients of P10,000 × g (c,d) and P135,000 × g (n = 4) (e,f) membrane pellets. (a,c,e) Concentrations of total protein (green, right Y axis) and formic acid (FA) extracted Aβ(1–40) (blue) and Aβ(1–42) (red) (left Y axis) measured by ELISA. (b,d,f) Corresponding in-vitro seeding (FRANK) assays of (b) differential centrifugation fractions from APP23 and wild-type mice (n = 3), or of (d,f) sucrose gradient fractions from P10,000 × g (d) and P135,000 × g (f) of APP23 (n = 4). Note that the FRANK assay contains 0.1 μg total proteins for each fraction. The lag times for fibrillation of 25 μM soluble Aβ(1–40) are shown (black: APP23, purple: WT) together with the assay concentrations of Aβ(1–40) (blue) and Aβ(1–42) (red). Data are presented as mean ± SEM. (g) Western blots with equal volumes of P10,000 × g and P135,000 × g sucrose gradient fractions detecting marker proteins of endoplasmic reticulum (Calnexin, sec22b), of lipid rafts (Flotilin-1), of lysosomes/late endosomes (Lamp2), of exosomes (CD63, Alix, Rab11) and of mitochondria (Tom20), as well as of two specific mitochondrial-associated ER membranes (MAMs) markers Mitofusin-2 (MFN2) and Fatty acid CoA ligase 4 (FACL4). Note that full-length blots/gels are presented in Supplementary Fig. 6.
