Recombinant ApoE2, ApoE3, and ApoE4 were produced both in HEK293 cells (FreeStyle 293-F cell, ThermoFisher) and bacteria (E. coli BL21 strain). Expression vectors for human ApoE2, ApoE3, and ApoE4 were constructed using an ApoE3 cDNA in pDONR221 vector (Gateway recombination system) that was obtained from Harvard Medical School (Harvard PlasmID Database, clone ID: HsCD00044600). Site-directed mutagenesis was used to generate ApoE2 (C526T) and ApoE4 (T388C) cDNAs. The ApoE2, ApoE3, and ApoE4 cDNAs were cloned into the lentiviral vector pLX304 (Addgene plasmid # 25890) using Gateway LR Clonase II (Invitrogen); the control plasmid was FUGW (Addgene plasmid # 14883), used to monitor transfection efficiency. 293-F cells were cultured in suspension in serum-free FreeStyle 293 Expression Medium (ThermoFisher, protein-free and chemically defined formulation) and transfected with control or ApoE expression plasmids using lipid-based FreeStyle MAX Reagent (ThermoFisher) following the manufacturer’s instructions. Supernatants from transfected 293-F cells were harvested 6 days after transfection and assessed by SDS-PAGE to determine the purity and yields of recombinant ApoE proteins (Fig. S1C). Stain-Free precast gels (Bio-Rad) were used for rapid fluorescent detection of proteins as an
