FIGURE 3.

Immunoblot analysis of SREBP-2 cleavage in Scap-deficient cells transfected with WT or Y640S mutant version of full-length Scap in the absence or presence of transfected Insig-1. A, Y640S mutant driven by TK promoter. On day 0, SRD-13A cells were set up for experiments at a density of 2.5 × 10⁵ cells/60-mm dish in 4 ml of medium A containing 5% FCS. On day 2, cells were transfected with 2 μg of pTK-HSV-BP2 and 0.4 μg of full-length WT pTK-Scap or 0.6 μg of its Y640S version with or without 0.2 μg of pTK-Insig1-Myc in 3 ml of medium A supplemented with 5% FCS; FuGENE 6 was used as the transfection agent. For each transfection, the total amount of DNA was adjusted to 2.8 μg/dish by the addition of pcDNA mock vector. On day 3, cells were washed once with PBS and then switched to hydroxypropyl-β-cyclodextrin-containing medium B for 1 h. The cells were washed with PBS and then incubated with medium C containing 30 μm cholesterol complexed with methyl-β-cyclodextrin as indicated. After incubation for 4 h, two pooled dishes of cells per condition were harvested, and the isolated nuclear and membrane fractions were subjected to immunoblot analysis with 0.167 μg/ml anti-HSV (SREBP-2), 1 μg/ml anti-Myc IgG-9E10 (Insig-1), or 5 μg/ml IgG-4H4 (Scap). B, Y640S mutant driven by CMV promoter. Experimental design was as in A except for DNA transfections. Cells were transfected with 2 μg of pTK-HSV-BP2 together with one of the following additional plasmids: none (lane 1); 0.4 μg of full-length WT pTK-Scap (lanes 2, 3, 8, and 9); 0.6 μg of mutant pCMV-Scap(Y640S) (lanes 4, 5, 10, and 11); or 0.4 μg of WT pTK-Scap plus 0.6 μg of mutant pCMV-Scap(Y640S) (lanes 6, 7, 12, and 13) with or without 0.2 μg of pTK-Insig1-Myc as indicated. For each transfection, the total amount of DNA was adjusted to 3.2 μg/dish by the addition of pcDNA mock vector. Films were exposed for 1–20 s.