Two AMPKKs can be resolved from rat liver extracts and both contain LKB1, STRADα and MO25α. (a) Separation of two activities that activate the GST-AMPKα1 catalytic domain by Q-Sepharose chromatography. The graph shows AMPKK activity in 4.5 ml fractions (red circles and red line), absorbance at 280 nm (continuous black line) and conductivity in the eluate (dashed black line) plotted against fraction number. (b) Probing of blots of column fractions after SDS gel electrophoresis (1 μl per lane) using anti-LKB1, anti-STRADα or anti-MO25α antibodies. In the three bottom panels, fractions 26–30 were concentrated from 4.5 ml to 250 μl using Amicon Ultra-4 30,000 MWCO centrifugal concentrators, and reanalyzed by western blotting using 2 μl per lane. (c) The effect of protein phosphatase treatment on the mobility of LKB1. The peak fractions of AMPKK1 (0.2 units) or AMPKK2 (0.8 units) were incubated in a final volume of 20 μl with or without 5 mM MgCl2 and 200 μM ATP for 15 min at 30°C. Protein phosphatases (PP1γ, 8 mU; or PP2A1, 1 mU) or buffer were added and incubation continued for a further 15 min before stopping the reactions in SDS sample buffer and analyzing by SDS gel electrophoresis and western blotting using anti-LKB1 antibody.