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.