Fig. 2

Phosphorylation of TET2 at Ser97 results in a stable TET2. a Mouse embryonic fibroblasts (MEFs) were serum starved for 14 h followed by treatment with 2-DG for 3 h. TET2 was detected by immunofluorescence staining (green). DAPI (4′,6-diamidino-2-phenylindole) was used to stain the cell nuclei (blue). b Wild-type or AMPK knockout mouse embryonic fibroblasts (MEFs) were treated with 100 µg/ml cycloheximide (CHX) alone, or in combination with AICAR for different periods. Levels of the indicated proteins are examined by immunoblotting analysis. GAPDH was used as a loading control. c Mutation of S97 to a non-phosphorylatable amino acid alanine (S97A) was sufficient to destabilize TET2. HEK293T cells were transfected with FLAG-tagged TET2 or FLAG-tagged TET2 S97A for 24 h; cells were then treated with 50 µg/ml CHX with or without 1 mM AICAR for different periods. Cell lysates were subjected to immunoblotting with the indicated antibodies. d Mutation of S97 to a glutamic acid that mimics phosphorylation of TET2 by AMPK results in a stable TET2. HEK293T cells were transfected with FLAG-tagged TET2, FLAG-tagged TET2-S97A or FLAG-tagged TET2-S97E for 24 h; cells were then treated with 50 µg/ml CHX for different periods. Cell lysates were subjected to immunoblotting with the indicated antibodies. e HEK293T cells were transfected with FLAG-TET2 for 24 h, then treated with 50 µg/ml cycloheximide (CHX) alone, or in combination with MG132 (10 µM), or Z-VAD-FMK (10 µM), or calpeptin (20 µM) for 12 h. Cell lysates were subjected to immunoblotting with the indicated antibodies. f 14-3-3β binds to phosphorylated TET2, but not to the S97A mutant. HEK293T cells were transiently transfected with the indicated constructs and treated with 1 mM AICAR for 3 h to induce AMPK activation. FLAG-TET2 was immunoprecipitated with M2 beads (Sigma), and Myc-14-3-3β was detected by Western blot analysis. g 14-3-3β binding stabilizes TET2. An increased amount of Myc-14-3-3β was co-transfected into HEK293T cells along with FLAG-TET2 or FLAG-TET2-S97A. Western blot analysis was performed to examine the expression of indicated proteins