This Article Abstract Full Text Services Email this article to a colleague Alert me to new issues of the journal Request Copyright Permission Citing Articles Citing Articles via CrossRef

Radhakrishnan et al. 10.1073/pnas.0700899104.

Supporting Information

Files in this Data Supplement:

SI Text

Reagents. We obtained [1,2,6,7-3H]cholesterol (60 Ci/mmol), [26,27-3H]25-hydroxycholesterol (75 Ci/mmol), [3-3H]lanosterol (10 Ci/mmol), and [1,2,6,7-3H]progesterone (100 Ci/mmol) from American Radiolabeled Chemicals; all other sterols from Steraloids; Fos-Choline 13 (n-tridecylphosphocholine) and Anapoe-C₁₂E₉ (polyoxyethylene(9)dodecyl ether/a-dodecyl-w-hydroxy-poly(oxy-1,2-ethanediyl) from Anatrace; phosphocholine chloride from Sigma; methyl-b-cyclodextrin (MCD) and hydroxypropyl-b-cyclodextrin (HPCD) from Cyclodextrin Technologies Development; Nonidet P-40 Alternative (NP-40) and digitonin from Calbiochem; and Fugene-6 reagent from Roche Applied Science. Lipoprotein-deficient serum and sodium oleate (1), delipidated serum (2), and solutions of sodium mevalonate and compactin (3) were prepared as described in the indicated reference.

Buffers. Buffer A contains 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM dithiothreitol, and 0.1% (w/v) Fos-Choline 13. Buffer B contains 50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1 mM dithiothreitol, and 0.1% (w/v) Anapoe-C₁₂E₉. Buffer C contains 50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM MgCl₂, 0.1% (w/v) SDS, 1.5% (w/v) NP-40, 0.5% (w/v) deoxycholate, and protease inhibitor mixture (25 mg/ml N-acetyl-leucinal-leucinal-norleucinal, 5 mg/ml pepstatin A, 2 mg/ml aprotinin, and 10 mg/ml leupeptin). Buffer D contains 10 mM Tris-HCl (pH 7.6), 100 mM NaCl, 1% SDS, and protease inhibitor mixture. Buffer E contains 50 mM Hepes-KOH (pH 7.6), 100 mM NaCl, 1.5 mM MgCl₂, 0.5% (w/v) digitonin, and protease inhibitor mixture.

Tissue Culture Media. Medium A is a 1:1 mixture of Ham's F-12 medium and Dulbecco's modified Eagle's medium containing 100 units/ml penicillin and 100 mg/ml streptomycin sulfate. Medium B is medium A supplemented with 5% (v/v) fetal calf serum. Medium C is Medium B supplemented with 5 µg/ml cholesterol, 1 mM sodium mevalonate, and 20 µM sodium oleate. Medium D is Medium A supplemented with 5% newborn calf lipoprotein-deficient serum, 50 mM sodium compactin, and 50 mM sodium mevalonate. Medium E is Schneider's Drosophila medium supplemented with 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin sulfate. Medium F is IPL-41 medium supplemented with 5% of heat-inactivated delipidated fetal calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin sulfate.

Expression Plasmids. The following plasmids were described in the indicated reference. pAc-Scap and pAc-Insig2-Myc encode hamster Scap and human Insig-2, each tagged with 6 copies of the Myc epitope tag at the COOH-terminus under control of the Drosophila actin SC promoter (4). pDS-HSV-SREBP2 encodes human SREBP-2 with an HSV tag at its NH₂-terminus under control of the Drosophila DS47 promoter (4). pTK-HSV-SREBP-2 encodes wild-type HSV-tagged human SREBP-2 under control of the weak thymidine kinase (TK) promoter (5). pCMV-Scap and pCMV-Insig2-Myc encode wild-type hamster Scap and human Insig-2, each tagged with 6 copies of the Myc epitope tag at the COOH-terminus under control of the cytomegalovirus (CMV) promoter (5). Mutants of Insig-2 in pAc-Insig2-Myc and pCMV-Insig2-Myc were generated with the QuikChange XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The integrity of all plasmids was confirmed by DNA sequencing of all open reading frames.

This Article Abstract Full Text Services Email this article to a colleague Alert me to new issues of the journal Request Copyright Permission Citing Articles Citing Articles via CrossRef