Extended Data Fig. 8: Validation steps of preparing the lipidated recombinant ApoE using POPC and cholesterol.

a. Technical replicates of recombinant ApoE3 or ApoE4 lipidated with POPC and cholesterol on an SDS-PAGE gel. ApoE was lipidated at a 1:50:10 molar ratio of ApoE:POPC:Cholesterol. SDS-PAGE was performed under nonreducing (NR) or reducing (R) conditions. Reducing conditions were performed with the addition of 50 mM DTT. One µg of protein was loaded in each well. SDS-PAGE was run on a 4–12% bis-tris gel with MES buffer. Gel was stained with coomassie blue showing only the presence of the ApoE protein. The y-axis represents molecular weight in kDA. Shown are three independent replicates of the samples. b. Native PAGE of technical replicates of recombinant ApoE3 and ApoE4 lipidated with POPC and cholesterol. ApoE was lipidated at a 1:50:10 molar ratio of ApoE:POPC:Cholesterol. One µg of protein was loaded in each well. High molecular weight ladder (Cytiva, 17044501) was detected using PonceauS staining. ApoE was detected by western blot using an anti-ApoE antibody (Academy Bio-Medical, 50A-G1b). Native gel shows the presence of lipidated ApoE. Shown are three independent replicates of the samples. c. FPLC curve of recombinant ApoE3 and ApoE4 lipidated with POPC and cholesterol. Lipidated ApoE was purified from fractions 15⁻20 and verified by negative stain TEM. Samples were run on a Superose 6 Increase 10/300 GL column (Cytiva, 29091596) at 0.5 mL/min in 20 mM phosphate buffer, 50 mM NaCl, pH 7.4. The x axis represents elution volume. d. Negative stain TEM of size exclusion chromatography fractions of recombinant ApoE3 and ApoE4 lipidated with POPC and cholesterol. Negative stain TEM imaging shows the presence of discoidal lipoprotein. Micrographs (N = 1/sample) were taken on a JEOL JEM-1400 at 120 kV at 80,000× magnification (0.138889 nm/pixel).