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33846649325
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note
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2O (9:1) to yield 2 (30 mg). Fraction 4 was purified by chromatography on silica gel (230-400 mesh, Merck, Ø50 × 150 mm), eluted with n-hexane/EtOAc (5:1) to yield 3 (70 mg).
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12
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33846602942
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note
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3, 300 MHz): δ 176.9, 175.4, 171.6, 168.5, 167.9, 147.4, 136.3, 133.2, 133.0, 131.3, 117.6, 111.7, 111.5, 69.7, 69.0, 46.4, 32.8, 29.2, 29.0, 25.7, 17.9.
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13
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0033084064
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14
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0141676293
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15
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33846567297
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note
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15. Briefly, Flp-In AC-29 cells containing five copies of integrated FRT sequence were generated by transfection of pFRT/lacZeo into AC-29 cells. FRT site-integrated clones were selected by 100 μg/mL zeocin (Invitrogen) for 1 week. Several colonies were picked and maintained in F-12 medium supplemented with 10% fetal bovine serum (FBS) and 100 μg/mL zeocin. The presence and number of FRT-site were verified by β-galactosidase assay and Southern blot analysis, respectively. To construct stable cell lines expressing hACAT-1 or hACAT-2, Flp-In AC-29 cells were plated on 60-mm dishes and transfected with 1.8 μg of the Flp-In recombinase-encoding pOG44 vector and either 0.2 μg of pcDNA5/FRT-hACAT-1 or pcDNA5/FRT-hACAT-2. After 2 days, we proceeded to select the single colony with 500 μg/mL hygromycin (Invitrogen) for 2 weeks. Colonies were picked up, expanded, and assayed for expression of hACAT-1 or -2. Established cell lines were maintained in F-12/10% FBS containing 100 μg/mL hygromycin.
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33846583836
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note
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15 a total of 30,000 cells per well were plated on 96-well culture plates and allowed to recover overnight. Assays were done with cells at least 80% confluent. Cells were incubated in Ham's F-12 medium supplemented with 1% Eagle's vitamins and 10% heat-inactivated FBS containing 5 μg/mL NBD-cholesterol as methyl-β-cyclodextrin complex with or without ACAT inhibitor for 9 h. NBD-cholesterol was added from a 5-mg/mL stock solution in methanol, and methanol concentrations in the medium did not exceed 0.1%. Flp-In AC-29 cells incubated with NBD-cholesterol were used to determine background fluorescence attributable to free NBD-cholesterol. After incubation, the medium was removed, and the cells were washed two times with cold balanced salt solution (BSS, Invitrogen). The fluorescent intensities of 96-well culture plates were read from the top using VICTOR3 fluorescent plate reader (Perkin-Elmer) equipped with 485 nm excitation and 535 nm emission filters. After measurement, cellular protein was digested through incubation with 25 μL of 0.4 N NaOH for 2 h. The fluorescent intensity was normalized by cellular protein content and calculated by subtracting the background fluorescent intensity from the total fluorescent intensity.
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33846588634
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note
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20 100 μM of MβCD was dissolved in 10 mL F-12 medium, and 6 μM NBD-cholesterol, and 10 μg/mL BSA were added to this solution with vigorous vortex. The dispersion was sonicated at room temperature for 5 min using a Fisher model 60 sonic dismembrator at setting 5. The solution was filtered with 0.45 μm membrane filter and kept in a glass tube under argon at 4 °C for up to a week.
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1242274634
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Lada A.T., Davis M., Kent C., Chapman J., Tomoda H., Ōmura S., and Rudel L.L. J. Lipid Res. 45 (2004) 378
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