S1 Two step mechanism of membrane disruption by Aβ through membrane fragmentation and pore formation Michele F.M. Sciacca 1 , Samuel A. Kotler 1 , Jeffrey R. Brender 1 , Jennifer Chen 1 , Dong-kuk Lee 1,2 , Ayyalusamy Ramamoorthy 1* 1 Biophysics and Department of Chemistry, University of Michigan, Ann Arbor, Michigan 48109- 1055 (USA) 2 Department of Fine Chemistry, Seoul National University of Science and Technology, Seoul, Korea 139-743 * Corresponding Author: Ayyalusamy Ramamoorthy 930 N. University Avenue, Ann Arbor, Michigan 48109-1055 (USA) Phone: +1 734 647-6572; Fax: +1 734 615-3790 E-mail: ramamoor@umich.edu Supporting Material: Additional Experimental Methods: Materials: Aβ 1-40 (97% purity) was purchased from Anaspec (Fremont, CA). 1-palmitoyl-2- oleoyl-sn-glycero-3-phosphocholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L- serine sodium salt (POPS), total ganglioside extract (Brain, Porcine-Ammonium Salt), and total brain lipid extract were purchased from Avanti Polar lipids Inc. (Alabaster, AL). Ferric chloride hexahydrate, ammonium thiocyanate, and Fura 2 pentapotassium salt were purchased from Sigma-Aldrich (St.Louis, MO). 6-Carboxyfluorescein was purchased from Fluka. Model membrane preparation. Large unilamellar vesicles (LUV) of POPC/POPS 7/3, POPC/POPS/ganglioside 5.5/3/1.5, and total lipid brain extract (TLBE) were prepared from a chloroform solution of lipids in the desired ratio. The solution was gently dried under nitrogen flow and then placed under a high vacuum overnight to further evaporate any residual solvent. The obtained lipid film was rehydrated with a buffer solution (10 mM phosphate buffer solution, pH 7.4, 100 mM NaCl), to yield a final concentration of 4 mg/ml, and dispersed by vigorous stirring. The resulting solution was extruded 23 times through a 100 nm polycarbonate Nucleopore membrane filter (Whatman) mounted on a mini-extruder in order to obtain LUV with an average diameter of 100 nm. To prepare dye-filled LUV, the dry lipid film was hydrated with a buffer solution containing 6-carboxyfluorescein (10 mM phosphate buffer solution, 70 mM 6- carboxyfluorescein, 100 mM NaCl, pH 7.4) or 100 μM Fura 2 pentapotassium salt (10 mM Hepes buffer solution, 200 µM EGTA, 100 mM NaCl, pH 7.4) to a final concentration of 10 mg/ml, which was followed by the procedures described above. Removal of any non- encapsulated 6-carboxyfluorescein or Fura 2 pentapotassium salt was performed by running the extruded LUV solution through a Sephadex G50 gel exclusion column (Sigma-Aldrich) and collecting the first colored band detectable under visible or UV light which contained the