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A 600-base pair probe from the haSR-BI cDNA (14) was used to isolate an mSR-BI clone from a murine 3T3-L1 adipocyte pcDNA1 library. GenBank accession number U37799; 89% amino acid identity and 96% similarity to haSR-BI (74) and 79% identity and 91% similarity to the human sequence CLA1 [D. Calvo and M A Vega, J. Biol Chem. 268, 18929 (1993)] The corresponding expression vector pmSR-BI-77 was transfected (14) into IdIA cells [D. M Kingsley and M. Krieger, Proc. Natl. Acad. Sci USA 81, 5454 (1984)] The receptor-positive cells, IdlA[mSR-BI] (done 15), were isolated by selection and flow cytometry (14).
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A 600-base pair probe from the haSR-BI cDNA (14) was used to isolate an mSR-BI clone from a murine 3T3-L1 adipocyte pcDNA1 library. GenBank accession number U37799; 89% amino acid identity and 96% similarity to haSR-BI (74) and 79% identity and 91% similarity to the human sequence CLA1 [D. Calvo and M A Vega, J. Biol Chem. 268, 18929 (1993)] The corresponding expression vector pmSR-BI-77 was transfected (14) into IdIA cells [D. M Kingsley and M. Krieger, Proc. Natl. Acad. Sci USA 81, 5454 (1984)] The receptor-positive cells, IdlA[mSR-BI] (done 15), were isolated by selection and flow cytometry (14).
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Acton, S.1
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4243059358
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note
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Cells were plated as in Fig. 1 in medium A (26) with 3% newborn calf lipoprotein-deficient serum. On day 2, cells were incubated with or without unlabeled HDL (20 μg of protein per militer for 5 hours at 37°C in medium B) and washed (Fig. 1), lipids were extracted twice with hexane: isopropanol (3:2; 3 ml for 30 min) Extracts were pooled, back-extracted with 1 ml of water, and dried, and total (free and esterified) cholesterol masses (averages of six replicates) were determined (Sigma Diagnostics, St Louis, MO). The protein contents were estimated from replicate cultures. The values of total cholesterol (micrograms per milligram of cell protein, ± SEM) for cells incubated without HDL were 20.5 ± 0.3 (IdlA) and 23.0 ± 0 4 (IdlA[mSR-BI]).
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note
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495.
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43
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4243189168
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unpublished results
-
The difference between the predicted mass of mSR-BI (57 kD) and the observed mass (82 kD) is due primarily to glycosylation (J. Babitt, A. Rigotti, M. Krieger, unpublished results).
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Krieger, M.3
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44
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note
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3H]cholesteryl oleate was extracted with isopropyl alcohol (30 min at room temperature) and quantified by scintillation counting. Dil was extracted with dimethyl sulfoxide (DMSO) and its fluorescence (550-nm excitation, 565-nm emission) measured with Dil-HDL standards dissolved in DMSO
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note
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We thank M. Chnstiansen and T. Hyatt for technical assistance and G. Baldini, H. Lodish, A Tall, P. Kim, L. Liscum, and members of our laboratones for materials and advice. Supported by NIH grants HL41484, HL52212, HL09047, and HL20948; The Perot Family Fund; and a Howard Hughes Medical Institute postdoctoral fellowship (A R.).
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