FIG. 4.

Expression of α1DN in hepatocytes. (A) Hepatocytes in culture were infected with varying titers of Ad.α1DN or Ad.null. Twenty-four hours after infection, cell lysates were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with an anti-α1 antibody. The α1-specific antibody used for detection recognizes both the endogenous α1 subunit and the recombinant α1 mutant protein. (B) Hepatocytes were infected with Ad.α1DN (10 PFU/cell), and at varying times after infection, AMPK complexes were isolated by immunoprecipitation (IP) with an anti-γ antibody. Proteins within the immune complex were resolved by SDS-PAGE, transferred to a PVDF membrane, and probed with either α2-, β-, or γ-specific antibodies. A control lane shows the expression of the subunits in an immune complex isolated from hepatocytes infected for 90 h with Ad.null (10 PFU/cell). (C) Hepatocytes were infected with either Ad.α1DN (10 PFU/cell) or Ad.null (10 PFU/cell). At various times after infection, total protein in cell lysates was analyzed for expression of α1 and the endogenous α2, β, and γ subunits, using subunit-specific AMPK antibodies. In each case, a representative blot from two independent experiments is shown.