Immunoblot analysis of SREBP-1 (A) and
SREBP-2 (B) in wild-type CHO-7 cells and mutant
25-hydroxycholesterol-resistant cells. On day 0, the indicated cell
line was set up for experiments as described. On day 2, cells were
switched to medium A containing 5% newborn calf lipoprotein-deficient
serum, 50 μM sodium mevalonate, and 50 μM compactin supplemented
with the indicated concentration of 25-hydroxycholesterol. After
incubation for 16 h, the cells were harvested and fractionated as
described, and aliquots of the nuclear extract (50 μg protein) and
membrane fractions (100 μg protein) were subjected to SDS/PAGE.
Immunoblot analysis was carried out with 30 μg/ml of IgG-2A4
(A) or 5 μg/ml IgG-7D4 (B) as described.
Filters were exposed to film for 30 sec (A Upper) or 5
sec (A Lower and B). P and M denote the
precursor and mature forms of SREBP-1 or SREBP-2, respectively.