Isolation of lipid rafts

For lipid raft fractionation from FAD hNPCs, cells were plated on thin-layer 3D matrix (1:10 Matrigel containing culture media), routinely used as a cellular model for AD (Kim et al., 2015). Lipid rafts were isolated from 0.5% Lubrol WX lysates of 10-day differentiated FAD hNPCs following published protocol (Bhattacharyya et al., 2013). To isolate ER-lipid rafts or MAMs, we first prepared crude Mitochondria fractions (Annunziata et al., 2013, 2018) prior to sucrose gradient centrifugation to isolate MAMs and non-MAMs. Briefly, 90% confluent cultured differentiated neuronal cells were resuspended in 5-volume homogenization buffer (10mM HEPES, pH 7.4, 0.25 M sucrose, protease inhibitors). The suspension was homogenized by 20-strokes in a glass homogenizer twice. Removing cell debris and nuclei after centrifugation (600 X g for 5min), the supernatant was subjected to sequential centrifugation steps as described before (Wieckowski et al., 2009) to isolate crude mitochondria (containing ER-rafts or MAMs) by discarding plasma membrane, lysosomes, microsomes and the cytosol. The crude mitochondria pellet was suspended in 0.5% Lubrol in 50 mM HEPES pH 7.4 containing 0.15 M NaCl, 5mM EDTA and protease inhibitor prior to sucrose gradient centrifugation as described before (Bhattacharyya et al., 2013). 1ml fractions were gently collected from the top of the gradient before immunoblotting with antibodies against MAM-proteins IP3R3 (anti-IP3R3) or ACAT (anti-ACAT) to determine MAM and non-MAM fractions.