Figure 2

AMPKK activity (that is, the ability to activate AMPKα1 catalytic domain), and LKB1, STRADα and MO25α polypeptides, can be immunoprecipitated from rat liver AMPKK1 and AMPKK2 using anti-LKB1 antibody. (a) Depletion of AMPKK activity from supernatant. Sheep anti-human LKB1 or pre-immune control immunoglobulin (IgG) was prebound to Protein G-Sepharose beads and cross-linked with dimethylpimelimidate as described [47], except that a final wash of the beads with 100 mM glycine, pH 2.5, was performed. Bead-bound antibodies (40 μl) were incubated with the peak fraction of AMPKK1 (0.04 units), AMPKK2 (0.03 units) or recombinant GST-LKB1:STRADα:MO25α complex (0.06 units) for 120 minutes and the beads removed in a microcentrifuge (14,000 × g for 2 min). AMPKK activity was determined in the supernatants and is expressed as a percentage of the value obtained using the control IgG. (b) The pellets from the experiment in (a) were resuspended in the original volume and samples of the supernatants and pellets analysed by western blotting with anti-LKB1 antibody. The recombinant LKB1 migrates at a higher molecular mass because of the GST tag. (c) As in (a), except that the amounts of AMPKK1, AMPKK2 and recombinant GST-LKB1:STRADα:MO25α complex were increased to 0.44, 0.70 and 1.4 units, respectively, and the activities were determined in the resuspended pellets. In this experiment the amount of antibody was limiting, so only a fraction of the activity was precipitated. (d) The pellets from the experiment in (c) were resuspended and samples analyzed by western blotting with anti-LKB1, anti-STRADα and anti-MO25α antibodies.