Figure 1.
Immunoblot analysis of SREBP-1 and SREBP-2 in wild-type CHO-7 and mutant 25-hydroxycholesterol-resistant cells. On day 0, the indicated cell line was set up in medium A supplemented with 5% lipoprotein-deficient serum. On day 2, the cells were switched to medium A containing 5% lipoprotein-deficient serum, 50 μM compactin, 50 μM sodium mevalonate, and 0.1% (vol/vol) ethanol containing the indicated final concentration of 25-hydroxycholesterol (25-OH Chol.). On day 3, the cells were harvested and fractionated into nuclear extract and 105 g membrane fraction as described (21, 29). Aliquots of the fractions (40 μg protein) were subjected to 7% SDS/PAGE and transferred to nitrocellulose. Immunoblot analysis was carried out with 10 μg/ml of IgG-2A4 (A) or 2 μg/ml of IgG-7D4 (B). Filters were exposed to film for 3 min (A, Upper), 45 s (A, Lower), 2 s (B, Upper), or 10 s (B, Lower). N and P denote the nuclear and precursor forms of SREBPs, respectively. X denotes a cross-reacting protein of unknown identity.