Isolation of rafts from mouse brain tissue by a detergent-free method
2008; Elsevier BV; Volume: 50; Issue: 4 Linguagem: Inglês
10.1194/jlr.d800037-jlr200
ISSN1539-7262
AutoresDixie‐Ann Persaud‐Sawin, Samantha Lightcap, G. Jean Harry,
Tópico(s)Lipid Membrane Structure and Behavior
ResumoMembrane rafts are rich in cholesterol and sphingolipids and have specific proteins associated with them. Due to their small size, their identification and isolation have proved to be problematic. Their insolubility in nonionic detergents, such as Triton-X 100, at 4°C has been the most common means of isolation. However, detergent presence can produce artifacts or interfere with ganglioside distribution. The direction is therefore toward the use of detergent-free protocols. We report an optimized method of raft isolation from lipid-rich brain tissue using a detergent-free method. We compared this to Triton-X 100-based isolation along sucrose or Optiprep™ gradients using the following endpoints: low protein content, high cholesterol content, presence of Flotillin 1 (Flot1), and absence of transferrin receptor (TfR) proteins. These criteria were met in raft fractions isolated in a detergent-free buffer along a sucrose gradient of 5%/35%/42.5%. The use of optiprep gave less consistent results with respect to protein distribution. We demonstrate that clean raft fractions with minimal myelin contamination can be reproducibly obtained in the top three low-density fractions along a sucrose step gradient. Membrane rafts are rich in cholesterol and sphingolipids and have specific proteins associated with them. Due to their small size, their identification and isolation have proved to be problematic. Their insolubility in nonionic detergents, such as Triton-X 100, at 4°C has been the most common means of isolation. However, detergent presence can produce artifacts or interfere with ganglioside distribution. The direction is therefore toward the use of detergent-free protocols. We report an optimized method of raft isolation from lipid-rich brain tissue using a detergent-free method. We compared this to Triton-X 100-based isolation along sucrose or Optiprep™ gradients using the following endpoints: low protein content, high cholesterol content, presence of Flotillin 1 (Flot1), and absence of transferrin receptor (TfR) proteins. These criteria were met in raft fractions isolated in a detergent-free buffer along a sucrose gradient of 5%/35%/42.5%. The use of optiprep gave less consistent results with respect to protein distribution. We demonstrate that clean raft fractions with minimal myelin contamination can be reproducibly obtained in the top three low-density fractions along a sucrose step gradient. Eukaryotic cell membranes consist of liquid-ordered states surrounded by liquid-disordered phases. This arrangement allows for the existence of small, organized membrane microdomains called rafts (1Brown D.A. London E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts.J. Biol. 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Their insolubility in nonionic detergents, such as Triton-X 100, Brij 96, Lubrol series, and Nonidet P40, at 4°C has enabled the isolation of raft-like structures termed detergent resistant membranes (DRMs) that have low buoyant densities and the ability to float on sucrose gradients. DRMS, like rafts, float away from detergent-soluble proteins and cytoskeletal proteins. The introduction of Iodixanol (OptiPrep™) provided researchers with an alternative to sucrose as a density gradient medium that is iso-osmotic up to a density of 1.32 g/ml (14Ford T. Graham J. Rickwood D. Iodixanol: a nonionic iso-osmotic centrifugation medium for the formation of self-generated gradients.Anal. Biochem. 1994; 220: 360-366Crossref PubMed Scopus (123) Google Scholar). Many studies have shown that some nonionic detergents fail to release DRMs at physiologically relevant temperatures (15Lingwood D. Simons K. Detergent resistance as a tool in membrane research.Nat. 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Isolation at physiological temperature of detergent-resistant membranes with properties expected of lipid rafts: the influence of buffer composition.Biochem. J. 2008; 417: 525-533Crossref Scopus (45) Google Scholar). Despite this there are caveats. Different detergents can yield varying subsets of DRMs, each with unique properties (19Arvanitis D.N. Min W. Gong Y. Heng Y.M. Boggs J.M. Two types of detergent-insoluble, glycosphingolipid/cholesterol-rich membrane domains from isolated myelin.J. Neurochem. 2005; 94: 1696-1710Crossref PubMed Scopus (61) Google Scholar, 20Heffer-Lauc M. Lauc G. Nimrichter L. Fromholt S.E. Schnaar R.L. Membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation.Biochim. Biophys. Acta. 2005; 1686: 200-208Crossref PubMed Scopus (62) Google Scholar, 21Heffer-Lauc M. Viljetic B. Vajn K. Scnaar R.L. Lauc G. 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Lipid rafts: contentious only from simplistic standpoints.Nat. Rev. Mol. Cell Biol. 2006; 7: 456-462Crossref PubMed Scopus (669) Google Scholar); cause abnormal redistribution of gangliosides; or alter raft properties (20Heffer-Lauc M. Lauc G. Nimrichter L. Fromholt S.E. Schnaar R.L. Membrane redistribution of gangliosides and glycosylphosphatidylinositol-anchored proteins in brain tissue sections under conditions of lipid raft isolation.Biochim. Biophys. Acta. 2005; 1686: 200-208Crossref PubMed Scopus (62) Google Scholar). The presence of detergents can also interfere with organelle and raft integrity (26Suneja S.K. Mo Z. Potashner S.J. Phospho-CREB and other phospho-proteins: improved recovery from brain tissue.J. Neurosci. Methods. 2006; 150: 238-241Crossref PubMed Scopus (5) Google Scholar), and may even produce anomalous or false-positive results, such as the unnatural oligomerization of amyloid-β (27Yu W. Zou K. Gong J.S. Ko M. Yanagisawa K. Michikawa M. Oligomerization of amyloid beta-protein occurs during the isolation of lipid rafts.J. Neurosci. Res. 2005; 80: 114-119Crossref PubMed Scopus (20) Google Scholar). Therefore, the general consensus is toward the use of detergent-free protocols. Such methods would provide the investigator not only with the option to use the isolated fractions for analyses where detergent presence would be detrimental, such as in proteomic or lipid analyses and screening (21Heffer-Lauc M. Viljetic B. Vajn K. Scnaar R.L. Lauc G. Effects of detergents on the redistribution of gangliosides and GPI-anchored proteins in brain tissue sections.J. Histochem. Cytochem. 2007; 55: 805-812Crossref PubMed Scopus (32) Google Scholar), but would also allow additional characterization of rafts. Most reports document the isolation of rafts from cultured cells (1Brown D.A. London E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts.J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2053) Google Scholar, 15Lingwood D. Simons K. Detergent resistance as a tool in membrane research.Nat. Protoc. 2007; 2: 2159-2165Crossref PubMed Scopus (223) Google Scholar, 28Brown D.A. Rose J.K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2605) Google Scholar, 29Schuck S. Honsho M. Ekroos K. Shevchenko A. Simons K. Resistance of cell membranes to different detergents.Proc. Natl. Acad. Sci. USA. 2003; 100: 5795-5800Crossref PubMed Scopus (553) Google Scholar). Although studying rafts in cell lines allows the investigator to examine the raft contribution from a single cell type, using tissue enables the examination of raft dynamics and proteolipid interaction within the organ system as a whole. The dynamic functions of lipid rafts have been reflected in the recent implication that they may be involved in the pathogenesis of many neurodegenerative conditions (30Ehehalt R. Keller P. Haass C. Thiele C. Simons K. Amyloidogenic processing of the Alzheimer beta-amyloid precursor protein depends on lipid rafts.J. Cell Biol. 2003; 160: 113-123Crossref PubMed Scopus (916) Google Scholar, 31Fantini J. Garmy N. Mahfoud R. Yahi N. Lipid rafts: structure, function and role in HIV, Alzheimers and prion diseases.Expert Rev. Mol. Med. 2002; 4: 1-22Crossref PubMed Scopus (171) Google Scholar, 32Persaud-Sawin D.A. McNamara 2nd, J.O. Rylova S. Vandongen A. Boustany R.M. A galactosylceramide binding domain is involved in trafficking of CLN3 from Golgi to rafts via recycling endosomes.Pediatr. Res. 2004; 56: 449-463Crossref PubMed Scopus (54) Google Scholar, 33Tashiro Y. Yamazaki T. Shimada Y. Ohno-Iwashita Y. Okamoto K. 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Raft aggregation with specific receptor recruitment is required for microglial phagocytosis of Aβ42.Glia. 2008; (In press.)Google Scholar, 31Fantini J. Garmy N. Mahfoud R. Yahi N. Lipid rafts: structure, function and role in HIV, Alzheimers and prion diseases.Expert Rev. Mol. Med. 2002; 4: 1-22Crossref PubMed Scopus (171) Google Scholar, 33Tashiro Y. Yamazaki T. Shimada Y. Ohno-Iwashita Y. Okamoto K. Axon-dominant localization of cell-surface cholesterol in cultured hippocampal neurons and its disappearance in Niemann-Pick type C model cells.Eur. J. Neurosci. 2004; 20: 2015-2021Crossref PubMed Scopus (25) Google Scholar, 35Harder T. Simons K. Caveolae, DIGs, and the dynamics of sphingolipid-cholesterol microdomains.Curr. Opin. Cell Biol. 1997; 9: 534-542Crossref PubMed Scopus (716) Google Scholar, 36Simons K. Gruenberg J. Jamming the endosomal system: lipid rafts and lysosomal storage diseases.Trends Cell Biol. 2000; 10: 459-462Abstract Full Text Full Text PDF PubMed Scopus (207) Google Scholar–37Simons K. Toomre D. Lipid rafts and signal transduction.Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39Crossref PubMed Scopus (5136) Google Scholar), and is therefore, the focus of this protocol. The majority of the work examining the role of rafts in the nervous system has relied on either primary cell cultures or isolated cellular preparations, such as from synaptosomes (38Igbavboa U. Eckert G.P. Malo T.M. Studniski A.E. Johnson L.N. Yamamoto N. Kobayashi M. Fujitia S.C. Appel T.R. Müller W.E. et al.Murine synaptosomal lipid raft protein and lipid composition are altered by expression of human apoE 3 and 4 and by increasing age.J. Neurol. Sci. 2005; 229–230: 225-232Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) and microvessicles (39McCaffrey G. et al.Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo.J. Neurochem. 2007; 103: 2540-2555Crossref PubMed Scopus (76) Google Scholar). This is due substantially to the large amount of lipid present in brain white matter in the form of myelin, which increases with age. For instance, at 6 months of age, 60 mg of myelin can be isolated from the rat brain, compared with only 4 mg at postnatal day (PND) 15 (40Siegel G.J. Molecular, cellular and medical aspects.in: Basic Neurochemistry. 6th edition. Lippincott-Raven, Philadelphia1998: 86-90Google Scholar). Thus, there should be a lower probability of myelin contamination of rafts isolated from PND 21 animals than from adult or older animals. By PND 21, myelin basic protein (MBP), a major myelin protein, has already been laid down in its mature form and is associated with raft fractions (41DeBruin L.S. Haines J.D. Bienzle D. Harauz G. Partitioning of myelin basic protein into membrane microdomains in a spontaneously demyelinating mouse model for multiple sclerosis.Biochem. Cell Biol. 2006; 84: 993-1005Crossref PubMed Google Scholar). MBP is involved in the maintenance of myelin and axonal integrity and may be associated with delayed-onset neurodegeneration (42Lappe-Siefke C. Goebbels S. Gravel M. Nicksch E. Lee J. Braun P.E. Griffiths I.R. Nave K.A. Disruption of Cnp1 uncouples oligodendroglial functions in axonal support and myelination.Nat. Genet. 2003; 33: 366-374Crossref PubMed Scopus (773) Google Scholar). It has also been used as an indicator of raft structural integrity (43Simons M. Krämer E.M. Thiele C. Stoffel W. Trotter J. Assembly of myelin by association of proteolipid protein with cholesterol- and galactosylceramide-rich membrane domains.J. Cell Biol. 2000; 151: 143-154Crossref PubMed Scopus (237) Google Scholar). Additionally, there is an age-dependent accumulation of ubiquinated 2',3′-cyclic nucleotide 3′-phosphodiesterase (CNP) in isolated myelin rafts (34Hinman J.D. Chen C.D. Oh S.Y. Hollander W. Abraham C.R. Age-dependent accumulation of ubiquitinated 2',3′-cyclic nucleotide 3′-phosphodiesterase in myelin lipid rafts.Glia. 2008; 56: 118-133Crossref PubMed Scopus (36) Google Scholar), which may alter their steady-state. We therefore chose to use PND21 mice as our model in this study, in lieu of adult mice. Regarding tissue protocols, raft-structures have been primarily isolated via detergent methods (19Arvanitis D.N. Min W. Gong Y. Heng Y.M. Boggs J.M. Two types of detergent-insoluble, glycosphingolipid/cholesterol-rich membrane domains from isolated myelin.J. Neurochem. 2005; 94: 1696-1710Crossref PubMed Scopus (61) Google Scholar, 23Chen X. Morris R. Lawrence M.J. Quinn P.J. The isolation and structure of membrane lipid rafts from rat brain.Biochimie. 2007; 89: 192-196Crossref PubMed Scopus (17) Google Scholar, 44Molander-Melin M. Blennow K. Bogdanovic N. Dellheden B. Månsson J.E. Fredman P. Structural membrane alterations in Alzheimer brains found to be associated with regional disease development; increased density of gangliosides GM1 and GM2 and loss of cholesterol in detergent-resistant membrane domains.J. Neurochem. 2005; 92: 171-182Crossref PubMed Scopus (189) Google Scholar). Although some use with nondetergent methods have been employed for brain cellular structures, these studies have used purified preparations (38Igbavboa U. Eckert G.P. Malo T.M. Studniski A.E. Johnson L.N. Yamamoto N. Kobayashi M. Fujitia S.C. Appel T.R. Müller W.E. et al.Murine synaptosomal lipid raft protein and lipid composition are altered by expression of human apoE 3 and 4 and by increasing age.J. Neurol. Sci. 2005; 229–230: 225-232Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar, 39McCaffrey G. et al.Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo.J. Neurochem. 2007; 103: 2540-2555Crossref PubMed Scopus (76) Google Scholar, 45Eckert G.P. Igbavboa U. Müller W.E. Wood W.G. Lipid rafts of purified mouse brain synaptosomes prepared with or without detergent reveal different lipid and protein domains.Brain Res. 2003; 962: 144-150Crossref PubMed Scopus (71) Google Scholar) and are not truly representative of brain tissue. Additionally, previously used protocols have multiple, long centrifugation and sonication steps that can result in the over-abundance of proteins not normally present within rafts and produce low yields. Another problem is the use of postnuclear supernatant (PNS) sources where both the source type and gradient can contain some form of detergent. MacDonald and Pike (48Macdonald J.L. Pike L.J. A simplified method for the preparation of detergent-free lipid rafts.J. Lipid Res. 2005; 46: 1061-1067Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) recently adapted detergent-free protocols from Song et al. (46Song K.S. Shengwen Li Okamoto T. Quilliam L.A. Sargiacomo M. Lisanti M.P. Co-purification and direct interaction of Ras with caveolin, an integral membrane protein of caveolae microdomains. Detergent-free purification of caveolae microdomains.J. Biol. Chem. 1996; 271: 9690-9697Abstract Full Text Full Text PDF PubMed Scopus (918) Google Scholar) and Smart et al. (47Smart E.J. Ying Y.S. Mineo C. Anderson R.G. A detergent-free method for purifying caveolae membrane from tissue culture cells.Proc. Natl. Acad. Sci. USA. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar) into a time-efficient method of raft isolation from cell line-derived PNS sources. Their protocol involves isolation of purified rafts by shearing cells in an isotonic buffer with cations, followed by separation along a 0% to 20% continuous OptiPrep™ gradient. This procedure has recently been used to isolate rafts from purified preparations of rat cerebral microvessels (39McCaffrey G. et al.Tight junctions contain oligomeric protein assembly critical for maintaining blood-brain barrier integrity in vivo.J. Neurochem. 2007; 103: 2540-2555Crossref PubMed Scopus (76) Google Scholar). Other detergent-free methods have used sodium carbonate and OptiPrep™; magnetic bead; or silica-based isolations using raft/caveolar proteins as markers or the use of cationic buffers, which may stabilize raft-associated proteins (47Smart E.J. Ying Y.S. Mineo C. Anderson R.G. A detergent-free method for purifying caveolae membrane from tissue culture cells.Proc. Natl. Acad. Sci. USA. 1995; 92: 10104-10108Crossref PubMed Scopus (675) Google Scholar, 48Macdonald J.L. Pike L.J. A simplified method for the preparation of detergent-free lipid rafts.J. Lipid Res. 2005; 46: 1061-1067Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar, 49Shah M.B. Sehgal P.B. Nondetergent isolation of rafts.Methods Mol. Biol. 2007; 398: 21-28Crossref PubMed Scopus (26) Google Scholar–50Liu J. Oh P. Horner T. Rogers R.A. Schnitzer J.E. Organized endothelial cell surface signal transduction in caveolae distinct from glycosylphosphatidylinositol-anchored protein microdomains.J. Biol. Chem. 1997; 272: 7211-7222Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). While these methods are useful for isolating rafts from isolated cells or cell structures, they have not been applied to whole tissue. Here, we employ the original MacDonald and Pike protocol (48Macdonald J.L. Pike L.J. A simplified method for the preparation of detergent-free lipid rafts.J. Lipid Res. 2005; 46: 1061-1067Abstract Full Text Full Text PDF PubMed Scopus (283) Google Scholar) as a starting point, and our optimization of this method for use on unprocessed brain tissue in the context of raft integrity. Calcium chloride, magnesium chloride, sucrose, sodium fluoride, sodium orthovanadate, phenylmethanesulphonylfluoride (PMSF), and protein inhibitor cocktail were obtained from Sigma-Aldrich (St. Louis, MO). SW60 ultracentrifuge tubes (#328874) were obtained from Beckman (Palo Alto, CA). BCA protein assay (#23227) and SuperSignal West Pico Chemiluminescent substrate were obtained from Pierce (Rockford, IL). Flotillin1 antibody (cat# 610821, BD Biosciences, Franklin Lakes, NJ), transferrin receptor antibody (TfR; cat# 13-6800, Zymed, San Francisco, CA), MBP antibody (cat# NBA-116, Assay Designs, Ann Arbor, MI), and Golgi reassembly and stacking protein 65 (GRASP65) antibody (cat# ab30315, Abcam, Cambridge, MA) were used for Western blots. Invitrogen (Carlsbad, CA) Western Breeze Chemiluminescent kits were used for Western blot protein detection. Optiprep™ was obtained from Axis-Shield (Norton, MA). The Cholesterol Assay Kit (#10007640) was obtained from Cayman Chemicals (Ann Arbor, MI). DMEM media, horse serum and FBS (entodoxin level < 0.1 EU/ml) were obtained from Invitrogen. PND 21 and adult (3 months old) C57BL/6 mice (Charles River Labs, Raleigh, NC) were anesthetized with CO2, decapitated, and brains excised. Cortical tissue from both hemispheres was dissected to obtain the cortex and underlying corpus callosum. The brainstem was also removed. One hundred milligram samples were immediately frozen on dry ice in microfuge tubes and stored at −80°C. Samples could be stored for a maximum of 1 year. All procedures were conducted according to an NIEHS Animal and Care Use Committee approved protocol. Frozen brain tissue samples were thawed on ice and homogenized in 500 μl of detergent-free lysis buffer [1× TBS (pH 8), 1% proteinase inhibitor cocktail, 1 mM PMSF, 5 mM NaF, 1 mM Na Orthovanadate] with the addition of 1 mM CaCl2, and 1 mM MgCl2 to render rafts more stable (23Chen X. Morris R. Lawrence M.J. Quinn P.J. The isolation and structure of membrane lipid rafts from rat brain.Biochimie. 2007; 89: 192-196Crossref PubMed Scopus (17) Google Scholar). The homogenate was sheared through a 23-gauge needle with 20 complete passes then centrifuged at 1,000 g for 10 min at 4°C and the PNS removed and maintained on ice. The process was repeated on the pellet. The final pellet was discarded unless required for comparison between cortex and brainstem tissues. The supernatants from both shearing steps were pooled and stored at −80°C for use later or immediately subjected to density gradient ultracentrifugation along sucrose or OptiPrep™ step density gradients. All steps were performed on ice and all reagents precooled to ≤4°C. Two hundred twenty-five microliters of the pooled supernatant was placed in pre-cooled SW60 ultracentrifuge tubes on ice and 225 μl of 85% sucrose/TBS mixed with gentle pipeting to prevent the formation of bubbles. To this, 3.0 mls of 35% sucrose/TBS was overlaid, followed by 675 μl 5% sucrose/TBS. This 5%/35%/42.5% gradient was compared with a 5%/20%/30% sucrose gradient. The tubes were centrifuged at 38,500 rpm (200,000 g) for 18 h at 4°C and compared with a 4 h spin time. Acceleration and deceleration rates were set to zero. After centrifugation, the mixture was clear except for a distinct, cloudy band at the interface between the 5% and 35% sucrose. Fifteen sequential fractions of 260 μl each were gently removed from the top of the tube and individually aliquoted. High-density fractions 14 and 15 were pooled as they are not normally considered as raft fractions (1Brown D.A. London E. Structure and function of sphingolipid- and cholesterol-rich membrane rafts.J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2053) Google Scholar, 10Persaud-Sawin D.A. Banach L. Harry G.J. Raft aggregation with specific receptor recruitment is required for microglial phagocytosis of Aβ42.Glia. 2008; (In press.)Google Scholar, 28Brown D.A. Rose J.K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface.Cell. 1992; 68: 533-544Abstract Full Text PDF PubMed Scopus (2605) Google Scholar). The fractions were stored at −80°C for a maximum of 6 months. To SW60 ultracentrifuge tubes, 225 μl of the pooled supernatant was added and mixed with 225 μl of 50% Optiprep™. To this, 3.0 mls of 20% Optiprep™ was overlaid, followed by 675 μl 10% Optiprep™. The tubes were centrifuged at 38,500 rpm (200,000 g) for 18 h at 4°C. Acceleration and deceleration rates were set to zero. Fifteen fractions of 260 μl each were gently removed from the top of the tube and individually aliquoted. High-density fractions 14 and 15 were pooled. The fractions were stored at −80°C for up to 6 months. While recent data identified confounding effects with detergent use, a significant amount of previous data on membrane rafts is based on the use of detergent-based isolation. We therefore compared isolation of rafts to the Triton-X 100 detergent method of preparing DRMs. All steps of the procedure were carried out with the tissue maintained on ice. Tissue was thawed on ice and homogenized in 500 μl of lysis buffer with detergent [1× TBS (pH 8), 1% Triton-X 100, 1% proteinase inhibitor cocktail, 1 mM PMSF, 5 mM NaF, 1 mM Na orthovanadate] and incubated on ice for 30 min. The homogenate was centrifuged at 1,000 g for 10 min at 4°C and the PNS removed and maintained on ice prior to loading on gradient within 10 min of removal. Total protein concentration was measured (below). From the supernatant, a 225 μl aliquot was subjected to ultracentrifugation for fractionation as described above using either (5%/35%/42.5) sucrose or OptiPrep™ as the gradient. The fractions were stored at −80°C for a maximum of 6 months. The total protein present in each lysate or per fraction was measured by a BCA protein assay (Pierce) using the microplate procedure as directed by the manufacturer. A 10 μl aliquot of each fraction was used for the protein assay. Sample absorbance was read at 540 nm. Protein concentrations ranged from 2–3 μg/ml. Fractions were thawed on ice and 50 μl of each fraction used to determine the amount of cholesterol according to manufacturer instructions (Cayman Chemicals). Total cholesterol was quantified as a fluorescent resorufin product measured at an emission wavelength of 590 nm using a microplate reader (Molecular Devices, Sunnyvale, CA). The Softmax Pro 4.3LS software was used for data collection (Molecular Devices). Lysa
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