Rabbits fed a diet containing 0.75% dihydrocholesterol for 7 days

On day 0, CHO-K1 cells were set up for experiments in medium B at 7 x 10^5 cells per 100-mm dish and cultured at 37°C. On day 2, the cells were switched to medium D containing 1% HPCD for 1 h. The cells were then washed twice with PBS and switched to medium D containing 20 mkM concentrations of Dihydrocholesterol complexed in a 1:12 molar ratio with MCD. Stock solutions of sterol–MCD complexes at a final sterol concentration of 2.5 mM. After incubation for 6 h, the cells were washed once with PBS, then treated with 300 mkl of Buffer D and scraped into 1.5-ml tubes. The cells were passed through a 22-gauge needle 15 times and then agitated for 10 min. The protein concentration of each cell extract was measured (BCA kit), after which an aliquot of cell extract (25 mkg) was mixed with an equal volume of Laemmli sample buffer (Bio-Rad), heated for 10 min at 95°C, and then subjected to 8% SDS/PAGE and immunoblot detection of SREBP-2.