Raft and non-raft cholesterol contents in Triton-Insoluble (TI) and Triton-Soluble (TS) membrane fractions isolated using 1% cold Triton (A) or Low Density (LD) and High Density (HD) membrane fractions isolated using non-detergent sonication protocol (B). Briefly, for both procedures, cells were scraped into buffer A (in mM): 150 NaCl, 20 HEPES, 5 EDTA, pH 7.4, 1x Protease Inhibitor Cocktail (PIC) (Roche, Indianapolis, IN), 1 μg/ml pepstatin, and homogenized in a Dounce tissue grinder (40 strokes), and centrifuged for 10 min at 1,000 g. The pellet was resuspended, dounced, and re-centrifuged for 10 min at 1,000 g. Combined supernatant was centrifuged for 1 h at 200,000 g to obtain the “high speed pellet” (SW40Ti rotor, Beckman Coulter, Fullerton, CA). For preparing Triton-soluble and Triton-insoluble fractions, the high-speed pellet was resuspended in 1 ml of Buffer A, sonicated 3×10 s, and supplemented with a small volume of concentrated solution of Triton X-100 to a final concentration of 1%. After 15 min incubation on ice, the suspension was centrifuged for 1 h at 200,000 g. Then, the pellet (Triton insoluble fraction) was resuspended in Laemmli buffer. For isolation of membrane fractions using non-detergent method, the total membrane pellet was resuspended in 1 ml of 45% sucrose solution, sonicated and layered on sucrose gradient (35%-5%). The sucrose gradient was then centrifuged for 18 hr at 100,000 g. After centrifugation, 11 fractions were collected, protein was precipitated by TCA and measured using BCA Protein Assay Kit (BioRad, Hercules, CA). The samples were then resolved on 12% SDS PAGE at reducing conditions followed by transfer to PVDF membranes.

Modulation of cellular cholesterol by MβCD:MβCD-cholesterol mixtures in Bovine Aortic Endothelial cells (A) and in Chinese Hamster Ovary Cells (B). Modified from Romanenko et al. (2002) [] and Byfield et al., []. MβCD saturated with cholesterol using the standard protocol. Briefly, small volume of a cholesterol stock solution in chloroform:methanol (1:1, vol:vol) was added to a glass tube and the solvent was evaporated. Then, 2.5 mM or 5 mM MβCD solution in DMEM medium without serum was added to the dried cholesterol. The tube was vortexed, sonicated, and incubated overnight in a shaking bath at 37°C. The mixture of MβCD and MβCD -cholesterol was prepared just by mixing the two solutions at different molar ratios.

Efficiencies of different sterol-cholesterol substitutions in Bovine Aortic Endothelial Cells. Modified from Romanenko et al., (2004) []. Briefly, the substitution experiments were performed by exposing the cells to MβCD solution saturated with a specific analogue. Different MβCD-sterol solutions were prepared as described above for MβCD-cholesterol.

A simplified scheme of effects of MβCD on lipid membranes The scheme illustrates complexity of MβCD interactions when used, as in a majority of studies, to disrupt membrane “rafts”. Cylinders represent MβCD, dark-headgroup phospholipids sphingomyelin, open circle-headroup lipids represent “other” phospholipids, e.g., PC. No 1:1 stoichiometry is implied (see text). A. MβCD approaches the membranes and captures cholesterol from both raft (1) and nonraft (2) regions. The mechanism of capture likely involves a membrane fluctuation-induced partial protrusion of cholesterol molecules into the aqueous phase. The preponderance of evidence indicates that the removal of cholesterol from raft domains is more efficient; thus short (2–10 min) exposures and/or low (≤ 1 mM) concentrations of MβCD may preferentially remove cholesterol from the raft regions. Longer exposures and higher MβCD concentrations may also lead to redistribution of cholesterol between the raft and nonraft regions (3,4). B. Cholesterol enrichment of membranes is originated by incubation of membranes with MβCD, previously loaded with cholesterol. Both raft (1) and nonraft (2) regions may increase their cholesterol content, and the relative degrees of enrichment are unclear. MβCD molecules upon delivering cholesterol to the membrane may bring on redistribution of cholesterol between the membrane regions (3,4), as well as redistribution of phospholipids (panel C). C. Prolonged exposures and high concentrations of MβCD may lead to removal of phospholipids from both raft (1) and nonraft (2) regions, as well as redistribution of different phospholipids between the regions (3).