Copyright information:Taken from "Complexes between the LKB1 tumor suppressor, STRADα/β and MO25α/β are upstream kinases in the AMP-activated protein kinase cascade"Journal of Biology 2003;2(4):28-28.Published online 24 Sep 2003PMCID:PMC333410.Copyright © 2003 Hawley et al., licensee BioMed Central Ltd. This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original URL. 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. 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. 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 PP2A, 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.