*4.4. Isolation and Fractionation of LDL-C*

Lipoproteins were isolated with sequential potassium bromide density ultracentrifugation as described [42]. The plasma was obtained from freshly collected whole blood samples, and 1% antibiotics (penicillin/streptomycin stock solution, Gibco), 0.5 mM EDTA (Thermo Fisher Scientific, Waltham, MA, USA), and a protease inhibitor cocktail (cOmplete, Roche, Sigma-Aldrich, St. Louis, MO, USA) were added to avoid ex vivo oxidation and degradation. LDL-C particles were isolated by using sequential potassium bromide density ultracentrifugation. Purified LDL-C was dialyzed against a degassed solution of 20 mM Tris-HCl and 0.5 mM EDTA at 4 ◦C with five buffer changes (once/day).

#### *4.5. Anion-Exchange Chromatography Purification of LDL-C Subfractions*

LDL subfractions were separated by using UnoQ12 anion-exchange columns (Bio-Rad Laboratories, Inc., Hercules, CA, USA) with an NGC Quest 10 chromatography system (Bio-Rad). The columns were pre-equilibrated with buffer A (0.02 M Tris-HCl (pH 8.0) and 0.5 mM EDTA) in a 4 ◦C cold room. After dialysis with buffer A, 100 mg of LDL in 10 mL was injected onto a UnoQ12 column and eluted at a flow rate of 2 mL/min with a multistep gradient of buffer B (1 M NaCl in buffer A). Five LDL fractions were eluted with a multistep gradient of buffer B according to electronegativity. L1 was the effluent collected between fractions 11 to 14 (18–28 min); L2, fractions 15 to 16 (28–32 min); L3, fractions 17 to 24 (32–48 min); L4, fractions 25 to 30 (48–60 min); and L5, fractions 31 to 40 (60–80 min). Protein concentrations were determined by using the bicinchoninic acid method. The respective fractions were then concentrated with Centriprep filters (YM-30, MilliporeSigma, Burlington, MA, USA) and sterilized by passage through 0.22 μm syringe filters.
