Fig. 2.

SIRT1 is O-GlcNAcylated in vitro and in vivo. a O-GlcNAcylation assays using recombinant human SIRT1 and OGT were performed in the presence or absence of UDP-GlcNAc. Reaction mixtures were immunoblotted with antibodies against O-GlcNAc (RL2), SIRT1, and OGT. Data represent two independent experiments. b Plasmids encoding Flag-SIRT1 and HA-OGT were transfected into NCI-H1299 cells, and the O-GlcNAcylation of SIRT1 was detected by either GlcNAz metabolic labeling with a biotin tag and probed with STV-HRP or the indicated antibodies. Data represent three independent experiments. c Detection of O-GlcNAcylated SIRT1 in the indicated cells by chemoenzymatic labeling. Cells were lysed, immunoprecipitated with antibody against SIRT1 and subsequently labeled with GalNAz and biotin, which were then probed with STV-HRP and antibody against SIRT1. Data represent two independent experiments. d Flag-tagged wtSIRT1 with or without HA-OGT was exogenously expressed in NCI-H1299 cells, and the cells were treated with or without 2 μM TMG for 4 h. Cells were lysed, immunoprecipitated with antibody against Flag-tag and subsequently labeled with GalNAz and biotin, which were then probed with STV-HRP and the indicated antibodies. Data represent three independent experiments. e NCI-H1299 cells, which were pretreated with 2 μM TMG for 4 h, were lysed and labeled with GalNAz and biotin. And then the biotinylated proteins were enriched and probed with indicated antibodies. Data represent two independent experiments