Figure 7.

Expression of C₂₇ 3β-HSD mRNA in wild-type and cholesterol 7α-hydroxylase (Cyp7a1) knockout mice fed low- and high-cholesterol diets. Poly(A)⁺-enriched mRNA was isolated from the livers (n = 5) of male and female wild-type mice (+) or cholesterol 7α-hydroxylase (Cyp7a1) knockout mice (–) fed normal chow containing 0.02% (wt/wt) cholesterol (Ctl) or chow supplemented with 1% (wt/wt) cholesterol for 21 days. Aliquots (5 μg) of this RNA were size fractionated with agarose-gel electrophoresis, transferred to nylon membranes, and subjected to blot hybridization using a radiolabeled probe derived from the mouse C₂₇ 3β-HSD cDNA. After washing, the filter was exposed to x-ray film for 72 hours. The location to which the C₂₇ 3β-HSD mRNA migrated in the experiment and its interpolated size (1.8 kb) are shown on the left of the autoradiogram. To ensure that equal amounts of mRNA were present in each lane of the membrane, it was stripped of radioactivity after the initial hybridization and rehybridized with a [³²P]-labeled probe derived from a rat cyclophilin cDNA. The filter was washed and exposed to x-ray film. The resulting autoradiogram, with a signal of equal intensity in each lane, is shown below that derived with the C₂₇ 3β-HSD cDNA probe. The calculated size of the cyclophilin mRNA (1.1 kb) is shown to the left of the autoradiogram.