FIGURE 9.

The α1-, α2-, γ1-, γ2-, and γ3-subunits of AMPK are O-GlcNAcylated. A, recombinant AMPK-α1β1γ1 or -α2β1γ1 complexes were incubated with recombinant O-GlcNAc transferase (ncOGT) in the presence of UDP-[³H]-GlcNAc [an autofluorograph ([³H]-GlcNAc; top panel) of the same gel stained with G250 Coomassie Blue (Total Protein; bottom panel)]. Reactions without AMPK (Ctrl) or ncOGT were included as negative controls. B, 12 possible AMPK heterotrimeric combinations of Myc-α1/2, β1/2, and γ1/2/3-FLAG were co-expressed in Hek293A cells and immunoprecipitated (IP) with anti-FLAG beads. IPs were immunoblotted (WB) for O-GlcNAcylated protein and AMPK-α. Anti-FLAG beads incubated without cell lysate (FLAG), and anti-FLAG IPs of cells expressing an empty (α) vector were included as negative controls. White boxes and black boxes outline bands corresponding to O-GlcNAcylated α- and γ-subunits, respectively. C, AMPK-α2 immunoprecipitates from Hek293A cells treated with vehicle (C) or GT (10 μm, 2 h) were immunoblotted for O-GlcNAcylated protein (CTD 110.6) and AMPK-α. Competition of CTD 110.6 antibody reactivity by 1 m GlcNAc (lower panel) confirms the specificity of the antibody for O-GlcNAcylated protein. The red box highlights where nonspecific CTD 110.6 reactivity of the α2-subunit would be. Primary antibody incubated without cell lysate (ab) and lysate incubated with nonspecific IgG (IgG) were included as negative controls. D, AMPK-α2 or -β2 immunoprecipitates of HeLa cells treated with vehicle (C) or GT (10 μm, 6 h) were immunoblotted for O-GlcNAcylated protein, AMPK-α and AMPK-β. The black box highlights O-GlcNAcylated α-subunit co-immunoprecipitated with β2. Primary antibody incubated without cell lysate (ab), and lysate incubated with nonspecific IgG (IgG) were included as negative controls.