Restoration of the ability of AMPK to be activated, and AMPK and acetyl-CoA carboxylase to be phosphorylated, by AICA riboside and phenformin in HeLa cells following expression of LKB1. Control HeLa cells (lanes 1,2,3), HeLa cells expressing wild-type LKB1 (WT; lanes 4,5,6) or kinase-inactive mutant LKB1 (D194A; KD, lanes 7,8,9) were incubated for 60 min with no further addition, with 2 mM AICA riboside or 10 mM phenformin, and lysed. (a) Endogenous AMPK was immunoprecipitated from the cell extracts and assayed. (b) The cell lysates was immunoblotted with antibodies recognizing AMPKα1 phosphorylated at Thr172 or total AMPKα1; the results were analyzed using the LI-COR Odyssey™ IR imager as described in the Materials and methods section, and are expressed as a ratio of the two signals. (c) The cell lysates were analyzed by western blotting and the membranes probed with antibodies recognizing ACC phosphorylated at Ser79, or streptavidin to determine total AMPKα1. The results were analyzed using the LI-COR imager as for (b).
Hawley et al. Journal of Biology 2003 2:28 doi:10.1186/1475-4924-2-28