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. Author manuscript; available in PMC: 2009 Apr 27.
Published in final edited form as: Mol Cell. 2008 Apr 25;30(2):214–226. doi: 10.1016/j.molcel.2008.03.003

Figure 5. AMPK Phosphorylation of Raptor induces 14-3-3 association.

Figure 5

A. Wild-type but not AA mutant raptor complexes with 14-3-3 only under energy stress conditions. HEK293 cells were co-transfected with pEBG or pEBG-14-3-3 with wild-type or AA mutant raptor then complexes were precipitated on glutathione beads. Beads or total cell extracts were immunoblotted with the indicated antibodies. Cells were treated with V, vehicle (DMEM) or P, 5mM phenformin for 1h.

B. Wild-type but not AA mutant raptor precipitates with recombinant GST-14-3-3 protein in extracts from energy stress treated TSC2−/− MEFs stably reconstituted with human raptor alleles. GST protein pulldowns or total cell extracts were immunoblotted with the indicated antibodies.

C. Endogenous raptor binds to immobilized recombinant GST-14-3-3 protein, but not recombinant GST protein, from extracts cells treated with energy stress in an LKB1-dependent manner. GST protein pulldowns or total cell extracts were immunoblotted with the indicated antibodies.

D. Myc-tagged raptor immunoprecipitates from phenformin (Ph) or vehicle (v) treated cells were eluted with myc peptide and immunoblotted for endogenous 14-3-3 isoforms as indicated.