Fig. 3 |. Glucose regulates TET2 stability via the TET2pS99 phosphorylation switch, which is controlled by AMPK.
a, Left, immunoblot signal of phosphoserine 99 (TET2pS99) on Flag–TET2WT immunoprecipitated from cells cultured in high or normal glucose. Right, quantification of TET2pS99 signals normalized by total TET2, *P = 0.027. b, LC–MS/MS validation of pS99 levels in the cells under high and normal glucose, *P = 0.030. c, Normalized TET2, TET2pS99 and pAMPK levels in PBMCs from healthy donors and patients with diabetes; n = 6, ****P = 1.01 × 10−6 (TET2), **P = 0.0035 (TET2pS99), ***P = 5.26 × 10−4 (pAMPK). d, Flag–TET2WT half-life and pAMPK levels under normal glucose or normal glucose switched to high glucose. e, Left, dot blot showing 5hmC levels in A2058-TET2WT cells after treatment with 100 μM A769662 (an AMPK activator). Right, normalized quantification of dot blot signals; *P = 0.029. f, CHX treatment to measure the half-life of Flag–TET2WT in cells treated with (+) or without (−) metformin (Met; 5 mM). g, Effects of knocking down AMPKα2 on the protein stabilities of Flag–TET2WT and TET2S99A. Scr, scrambled shRNA. h, Half-lives of Flag–TET2S99D and TET2S99A. i, HPLC–MS/MS results showing the effects of knocking down AMPKα2 on 5hmC levels in TET2WT (**P = 0.006 (sh1), 0.003 (sh2)) cells and cells overexpressing TET2S99A or TET2S99D. Data represent three biologically independent repeats in a, b, d–i. Two-sided Student’s t-test, data shown as mean ± s.d., * P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. For gel source data, see Supplementary Fig. 1.