(A) CYP7A1 and cyclophilin mRNA levels in livers (n = 9) of wild-type and Shp−/− mice were determined by blot hybridization of poly(A)+-enriched RNA. Densitometric scanning of the resulting autoradiogram indicated that the CYP7A1 mRNA was 2- to 3-fold more abundant in knockout mice.
(B) Enzyme activity was measured in a gas chromatography-based assay using hepatic microsomes isolated from animals (n = 5) of the indicated Shp genotypes. Values in the mutant mice were approximately 20%–30% higher than those in the controls.
(C) Immunodetection of CYP7A1 protein. Aliquots containing approximately 150 μg of microsomal protein were subjected to immunoblotting as described in Experimental Procedures. Signals emanating from Shp−/− samples were approximately 2-fold stronger as judged by densitometry, while those from a control protein (heavy chain binding protein, BIP) were unchanged.