Time course of 6PF-2-K/F-2,6-P2ase overexpression in the liver of normal mice treated with Ad-Bif-DM. (a) Western blot analysis of 6PF-2-K/F-2,6-P2ase. 6PF-2-K/F-2,6-P2ase was extracted from fresh liver of normal mice on day 0 (without adenovirus infusion, lanes 1 and 2), day 3 (lanes 3 and 4), day 5 (lanes 5 and 6), and day 7 (lane 7 and 8), from mice that had received Ad-Bif-DM infusion. Concentration of extracted protein was measured by the BCA method. Equal amounts of extracted protein (100 μg) were loaded in each lane for SDS-PAGE and transferred to PVDF membrane by electrophoresis. Anti-rat liver 6PF-2K/F-2,6-P2ase polyclonal antibody served as primary antibody at 1:1,000 dilution. (b) A total of 50 or 100 μg extracted protein was used for kinase activity assay by catalyzing synthesis of F-2,6-P2 from 25 mM F-6-P.