Fig. 4

AKTi induces FOXO3a acetylation by disrupting the SirT6/FOXO3a interaction. a Flag-tagged Sirt family members were co-expressed with HA-FOXO3a in HEK293 cells. After IP with HA antibody, the bound Sirt members were examined by western blotting using anti-Flag antibody. b After BT474 and T47D cells were treated with various AKTi for 4 day, cell extracts were subjected to IP with FOXO3a antibody, binding of endogenous SirT6 was analyzed by western blotting. c Expression of endogenous SirT6 was knocked down by siRNA in BT474 cells, followed by treatment with 1 μM MK2206 for 2 days. After IP with FOXO3a antibody, endogenous FOXO3a acetylation was analyzed by western blotting using pan acetylated-lysine antibody. d Acetylated FOXO3a was purified from TSA and Nicotinamide-treated BT474 cells by FOXO3a antibody and then incubated with purified recombinant SirT6 protein in a deacetylation assay as described in the Experimental Procedures. The acetylated state of FOXO3a was assessed by western blotting using pan acetylated-lysine antibody. The immuno-purified FOXO3a and SirT6 used in this assay were analyzed by western blotting. e WT or TM mutant (with T32, S253, and S315 of FOXO3a changed to alanine, which confers FOXO3a localized exclusive in the nucleus) of FOXO3a were expressed in HEK293 cells followed by treatment with 1 μM MK2206. After IP FOXO3a, the association of SirT6 was analyzed by western blotting using anti-Flag antibody. f BT474 and T47D cells were pre-treated with 1 μM MK2206 followed by stimulation with 10 ng/ml of IGF-1 for 4 h, cell extracts were subjected to IP with SirT6 antibody, phosphorylation of endogenous SirT6 was analyzed by either specific SirT6-S338 phosphorylation or phospho-AKT substrate antibody by western blotting, respectively. g BT474 and T47D cells were treated with 1 μM MK2206, cell extracts were subjected to IP with AKT antibody, the bound SirT6 was analyzed by western blotting. h WT, S338A, or S338E mutant of SirT6 were expressed in HEK293 cells followed by treatment with or without 1 μM MK2206. After IP FOXO3a, the bound SirT6 was analyzed by western blotting using anti-Flag antibody