Fig. 3.

Effects of sterols on SREBP-2 cleavage. On day 1, WT cells were fed with medium A containing 5% (vol/vol) LPDS, 1 mM mevalonate, 20 μM oleate, and the indicated sterols at 3 μg/ml (final concentration of 0.3% ethanol for all media). Cells were refed every 2 days thereafter. (A) Cells were fixed and stained on day 5. (B) Cells were fixed and stained on day 10. (C)On day 0, WT cells were set up in medium A at 500,000 per 10-cm dish. On day 2, cells were refed inducing medium [medium A containing 5% (vol/vol) LPDS, 50 μM compactin, and 50 μM mevalonate (17)] or the same medium further supplemented with 10 μg/ml cholesterol and a 2 μg/ml concentration of the indicated sterol. All media were adjusted to 0.3% ethanol. After 4.5 h, N-acetyl-leucinyl-leucinyl norleucinal (ALLN) was added to a final concentration of 50 μg/ml. Cells were harvested 90 min later and lysed in SDS buffer [10 mM Tris·HCL, pH 7.6/100 mM NaCl/1% SDS containing protease inhibitors pepstatin A (5 μg/ml), lupeptin (10 μg/ml), ALLN (25 μg/ml), aprotinin (1,000 units/ml), and phenymethylsulfonyl fluoride (0.5 mM)]. Aliquots (50 μg of protein) were analyzed by SDS/PAGE and Western blotting with 5 μg/ml monoclonal antibody 7D4 against hamster SREBP-2 (39). P, membrane-bound SREBP-2 precursor; N, cleaved, nuclear SREBP-2.