Extended Data Fig. 4 |. Purification of full-length TET2 recombinant proteins and determination of TET2 S99 phosphorylation by AMPK.

a, SDS–PAGE gel showing the quality and quantity of recombinant TET2WT, TET2S99A, TET2SLF and TET2S1205A proteins. b, Representation of TET2 S99 phosphorylation levels, as detected by LC–MS/MS after in vitro kinase assay. The peak areas represent the abundance of peptides containing non-phosphorylated S99 (lower lane) or phosphorylated S99 (upper lane). The levels of S99 phosphorylation were much higher after the addition of AMPK in the in vitro kinase assay. c, Various amounts of unphosphorylated or phosphorylated TET2S99 peptides were spotted onto nitrocellulose membrane and detected by purified anti-TET2 pS99 antibody (right) or unpurified whole serum (left). The TET2pS99 antibody specifically recognized phosphorylated S99 peptides. Figures in a–c represent three biologically independent repeats each. d, Quantification of the radioactivity of AMPK phosphorylation. At the end of the phosphorylation assay, 20 μg of full-length Flag–TET2 WT and mutant proteins bound to beads were subjected to measurements by Liquid Scintillation Analyzer. The calculation is based on the specific activity of [γ−³²P]ATP (800 c.p.m. per pmol) used and the calculated molar amounts of TET2 added to the assay tube. The stoichiometry is around 0.38 mol phosphates per mole of TET2WT. This number decreases markedly if the TET2 protein is mutated (S99A mutant or SLF mutant, both of which disrupt the AMPK-recognizing consensus sequence). TET2WT and the S1205A mutant had comparably high levels of phosphorylation. n = 3 biologically independent repeats, with data shown as mean ± s.d.