Recombinant LKB1:STRAD:MO25 complexes can efficiently activate the AMPKα1 catalytic domain via phosphorylation at Thr172. (a) The indicated combinations of GST-tagged wild-type LKB1 (WT, lanes 1–9), or kinase-dead (D194A; KD, lanes 10–13) LKB1 mutant, or GST-alone (lane 14), FLAG-tagged STRADα or STRADβ, and Myc-tagged MO25α or MO25β were coexpressed in HEK-293T cells, purified on glutathione-Sepharose and tested for their ability to activate GST-AMPKα1 catalytic domain (top panel). The results are expressed as the increase in the units of AMPK activity generated per mg full-length GST-AMPKα1 catalytic domain. Samples from each incubation were also analyzed by western blotting and probed using the indicated antibodies (from top to bottom): anti-pT172; anti-AMPKα1 catalytic domain (GST-AMPKα1); anti-GST to detect GST-LKB1; anti-FLAG to detect STRADα and STRADβ, and anti-Myc to detect MO25α and MO25β. All proteins migrated with the expected mobility, taking into account the epitope tags. The bottom three blots were conducted on blank reactions lacking GST-AMPKα1 catalytic domain, as the latter appeared to cause some interference with detection. (b) Recombinant GST-LKB1:STRADα:MO25α complex was used to phosphorylate wild-type GST-AMPKα1 catalytic domain (GST-α1-WT) or a T172A mutant (GST-α1-T172A) using [γ-32P]ATP as described in Materials and methods. The reaction was analyzed by SDS gel electrophoresis and autoradiography. Arrows show the migration of GST-LKB1 (which autophosphorylates) and GST-AMPKα1 catalytic domain.