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Sun et al. 10.1073/pnas.0700907104.

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Reagents. We obtained Anti-Flag M2 Affinity Gel (catalog no. A2220) from Sigma; Protein A/G beads from Santa Cruz Biotechnology; digitonin and Nonidet P-40 from Calbiochem; mPEG-MAL-5000 (methoxy poly(ethylene glycol) maleimide; catalog no. 2E2M0H01) from Nektar Therapeutics (Huntsville, AL); 25-hydroxycholesterol (25-HC) and other sterols from Steraloids; 2-hydroxypropyl-b-cyclodextrin (HPCD) and methyl-b-cyclodextrin (MCD) from Cyclodextrin Technologies Development, Inc. Solutions of sodium mevalonate and compactin were prepared as described (1, 2). Newborn calf lipoprotein-deficient serum (LPDS) (d>1.25 g/ml) was prepared by ultracentrifugation (2). All other reagents were obtained from reported sources (3).

Antibodies. The following antibodies were described in the indicated reference: polyclonal IgG-R139 against hamster Scap (amino acids 54-277 and 540-707) (4), monoclonal IgG-9D5 against hamster Scap (amino acids 540-707) (4), and monoclonal IgG-9E10 against C-Myc (5). We obtained monoclonal IgG-GM130 against the Golgi protein GM130 (catalog no. 610822) from BD Transduction Laboratories; monoclonal anti-HSV IgG (69171-4) and monoclonal anti-T7 IgG (69522-4) from Novagen; Alexa Fluor 594 goat anti-mouse IgG (A-11032), Alexa Fluor 594 goat anti-rabbit IgG (A-11037), and Alexa Fluor 350 goat anti-rabbit IgG (A-21068) from Molecular Probes. Polyclonal rabbit anti-MELADL antibody was generated against a 16-amino acid synthetic peptide corresponding to amino acids 446-461 of hamster Scap (6). The peptide, RMELADLNKRLPPESC (synthesized by the Protein Chemistry Technology Center at the University of Texas Southwestern Medical Center at Dallas), was conjugated to Keyhole limpet hemocyanin using the Imject Maleimide Activated mcKLH Kit (Pierce); mixed with complete Freund's adjuvant; and injected into rabbits (7). The anti-MELADL antibody was affinity-purified using the unconjugated synthetic peptide immobilized on SulfoLink Coupling Gel (Pierce) according to the manufacturer's instructions.

Fab fragments of affinity-purified anti-MELADL and IgG-R139 were produced by incubation with immobilized papain using the ImmunoPure Fab Preparation Kit (Pierce). The Fab fragments used for microinjection of cells were concentrated with a Microcon Centrifugal Filter Device (YM-50; Millipore) and diluted into buffer A (10 mM Hepes-KOH at pH 7.2 and 50 mM potassium acetate) at a final concentration of 0.2 mg/ml.

Expression Plasmids. The following previously characterized plasmids were described in the indicated reference: pCMV-Scap, which encodes the open reading frame of hamster Scap under control of the cytomegalovirus (CMV) promoter (8); pCMV-Insig1-Myc and pCMV-Insig1-T7, which encode human Insig-1, followed by six tandem copies of a Myc tag and three tandem copies of a T7-epitope tag (MASMTGGQQMG), respectively (9, 10); and pTK-HSV-BP2, which encodes full-length human SREBP-2 with a HSV tag at its NH₂-terminus under control of the thymidine kinase (TK) promoter (4).

pCMV-Scap(1-767;Cys-), which encodes the membrane domain of hamster Scap(1-767) with alanine substitutions for cysteine at five residues (22, 461, 728, 734, and 755) and for proline at one residue (463), was constructed using the QuickChange II XL Site-directed Mutagenesis Kit (Stratagene). Using pCMV-Scap(1-767;Cys-) as a template, we also generated two mutant plasmids in which one residue in each plasmid (M447 or R445) was changed to cysteine by site-directed mutagenesis, i.e., pCMV-Scap(1-767;Cys-;M447C) and pCMV-Scap(1-767;Cys-;R445C). The coding regions of all plasmids were verified by DNA sequencing.

Tissue Culture Media. Medium A contains a 1:1 mixture of Ham's F-12 and Dulbecco's Modified Eagle's Medium supplemented with 100 mg/ml streptomycin sulfate and 100 units/ml penicillin. Sterol-depleting medium contains medium A supplemented with 5% (v/v) newborn calf lipoprotein-deficient serum, 50 mM sodium mevalonate, and 10 mM sodium compactin. Microinjection medium contains Dulbecco's Modified Eagle Medium (4.5 g/L D-glucose; 12100-061, Invitrogen) buffered with 25 mM Hepes-KOH at pH 7.2, supplemented with 100 µg/ml streptomycin sulfate and 100 units/ml penicillin. Sterol-depleting imaging medium contains CO₂-Independent Medium (18045-088, Invitrogen) supplemented with 2 mM glutamate, 5% lipoprotein-deficient serum, 1% HPCD, 50 µM sodium compactin, 50 µM sodium mevalonate, 100 µg/ml streptomycin sulfate, and 100 units/ml penicillin.

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