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 alternative to Coomassie staining. Upon UV illumination, the trihalo compounds contained in Stain-Free gels covalently bind to tryptophan residues of analyzed proteins and generate fluorescence. After centrifugation of ApoE2, ApoE3, and ApoE4 for 4 hours at 50,000g, more than 90% of the ApoE remained in the supernatant with apparently equal amounts of ApoE2, ApoE3, and ApoE4. The ApoE supernatants were diluted to proper concentration by fresh NBA/B27 (typically 50–80X) and added into cultured neurons for ApoE stimulation. The supernatant from control- (FUGW-) transfected 293-F cells was diluted the same way and added onto neurons as the control condition, in order to control the possible influences from other HEK293-secreted proteins. HEK293 ApoE proteins were used in all ApoE stimulation experiments unless otherwise indicated.
