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. 2011 Nov 7;286(52):44532–44541. doi: 10.1074/jbc.M111.315895

FIGURE 8.

FIGURE 8.

Human AMPK α-hook domain is required for adenylate-mediated protection in vitro. A, structural model for human AMPK in the active conformation (31) showing the α subunit (green), with its α-hook domain (red sticks) reaching into site 3 of the γ subunit (cyan) bound to ADP (blue). B, Coomassie Blue-stained protein gel showing the purity of the recombinant wild-type AMPK α1β1γ1 complex and the same complex with the α-hook deleted (AMPK d377–411). The mobility of molecular mass markers (M) is indicated in kilodaltons on the left. C, phosphatase protection assay with AMPK heterotrimers with and without the α-hook domain. Triplicate reactions were treated with PP2C with or without 0.2 mm ADP as shown. Mean values of the percentage of phosphorylated AMPK (AMPK-P) to total AMPK remaining after phosphatase treatment are plotted. Representative blots are shown below. D, titration of PP2C using wild-type AMPK (□) or AMPKΔ377–411 (●). The percentage of phosphorylated AMPK to total AMPK remaining after phosphatase treatment is plotted as a function of added PP2C. ***, p < 0.001; ns, p > 0.05.