The following samples were used to detect glycosylation of the indicated proteins in Fig. 1c: 293T whole cell lysate (Sp1), rat hypothalamus crude nuclear pellet (MeCP2), rat brain detergent-soluble fraction (synapsin IIa, Nup62), whole cell lysate from cultured embryonic neurons (CREB), cytosolic fraction from PUGNAc-treated cultured embryonic neurons (OGA), and p75-OGT purified from Sf9 cells. For Fig. 3a, the liver, hippocampus or cerebral cortex was harvested from adult Sprague Dawley rats, and crude nuclear pellets were processed. Animal protocols were approved by the Institutional Animal Care and Use Committee at Caltech, and the procedures were performed in accordance with the Public Health Service Policy on Humane Care and Use of Laboratory Animals. Cell lysates were prepared as described in the Supplementary Methods. Each sample and its corresponding negative control (lacking ketogalactose probe 1 incorporation) was subjected to chemoenzymatic labeling with PEG mass tags, resolved on 4–12% Bis-Tris NuPAGE gels (Invitrogen), and transferred to nitrocellulose or PVDF membranes. The membranes were immunoblotted with antibodies against each protein of interest (see Supplementary Methods). After incubation with secondary antibodies (IRDye 800 goat anti-rabbit or Alexa Fluor 680 goat anti-mouse), proteins were visualized and quantified using an Odyssey infrared imaging system (LI-COR Biosciences). To quantify O-GlcNAc stoichiometries, the intensities of the PEG-shifted band (glycosylated protein fraction) and the unshifted band (non-glycosylated protein fraction) were measured using Odyssey imaging software (Version 2.1). The resulting values of the PEG-shifted bands were corrected for non-specific background by subtracting the background intensity from negative control reactions. For data and statistical analyses, mean values, standard error of the mean, and P-values (paired, two-tailed, Student’s T-tests, α-value = 0.05) were calculated using the program Excel.