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. 2004 Feb 19;23(4):833–843. doi: 10.1038/sj.emboj.7600110

Figure 8.

Figure 8

AICAR does not activate AMPK-related kinases. (A) LKB1+/+ MEFs were either left untreated or stimulated with the indicated concentrations of phenformin (Phen) for 1 h. AMPKα1/α2 and LKB1 were immunoprecipitated from the cell lysates, and in vitro kinase activity towards the AMARA and LKBtide peptides, respectively, was measured as described in Materials and methods. Results shown are average±s.d. of a triplicate assay and are representative of two independent experiments. (B) To confirm equal expression of the kinases in each sample, cell lysates were subjected to SDS–PAGE and Western blot analysis with the indicated antibodies. The phospho-Thr172 antibody (P-AMPK) recognises the phosphorylated T-loop of AMPKα1. (C) LKB1+/+ MEFs were either left untreated (black bars) or stimulated with 2 mM AICAR (white bars) for 1 h. AMPKα1 and the indicated AMPK-related kinases were immunoprecipitated from the cell lysates, and in vitro kinase activity towards the AMARA peptide was measured as described in Materials and methods. Results are presented as % relative to the activity observed in nonstimulated cells, and are averages±s.e.m. of two independent experiments. 100% corresponds to the following absolute activities: AMPKα1, 135 mU/mg; NUAK2, 0.25 mU/mg; QIK, 0.76 mU/mg; QSK, 1.9 mU/mg; SIK, 2.73 mU/mg; MARK1, 0.14 mU/mg; MARK2/3, 3.5 mU/mg; and MARK4, 1.6 mU/mg. (D) Immunoblotting of AMPK was performed as in (B).