Effect of knockdown of the α2 isoform of AMPK on the activity of the AMPK pathway in MPT cells from α 1
and WT mice. MPT cells from α1-/- mice, or their WT control, were infected with control shRNA or anti-α2 shRNA designed to knock down the expression of the α2 isoform of AMPK. MPT cells were subjected to metabolic stress by incubation in medium containing dextrose (5 mM) plus antimycin (2 μM) for 4 hrs. Control cells were incubated in dextrose in the absence of antimycin. Activation of the AMPK pathway was assessed by immunoblotting using antibodies that recognize either the phosphorylated (activated) form of AMPK or the phosphorylated (inhibited) form of ACC. Expression of total AMPK was also assessed. Upper panel: Representative immunoblot. Molecular weight markers (in kDa) are shown om the right of each immunoblot. Lower panel: Densitometric quantitation of three immunoblots using β-actin as a loading control. * p < 0.01, anti-α2 shRNA vs. control shRNA, for total α domain expression in MPT cells from WT or α1-/- mice; # p < 0.02, anti-α2 shRNA vs. control shRNA, for phospho-AMPK expression in MPT cells from WT or α1-/- mice; p < 0.02, anti-α2 shRNA vs. control shRNA, for phospho-ACC expression in MPT cells from WT or α1-/- mice.