Figure 4.

Specificity of the mitochondrial targeting of P4501A1 under in vivo conditions. In lanes marked T, COS cells were transfected with full-length 1A1 construct (10 μg/plate) and cotransfected with 10 μg/ plate of the human P450 reductase cDNA and 5 μg/ plate of bovine Adx cDNA constructs. In lanes marked M, cells were mock transfected with equivalent amount of pCMV-4 plasmid DNA. Mitochondrial and microsomal fractions were isolated as described in Materials and Methods, and 20 μg of microsomal and 30 μg of mitochondrial proteins were resolved on a 14–16% gradient polyacrylamide gel and were subjected to Western blot analysis using the indicated antibodies. (A and C) Mitochondrial and microsomal fractions in duplicate lanes were derived from two separate transfections. (B) Microsomal and mitochondrial proteins were digested with 100 μg/ml (+) or 200 μg/ml of reaction (++) with pronase for 30 min on ice before electrophoresis.