Figure 1.
Preparations enriched in ribosomal components contain numerous O-GlcNAc proteins. (A) Postmitochondrial (PM), postribosomal (PR), and ribosomal (Ribo) preparations were obtained by subcellular fractionation of rat pancreas and subjected to galactosyltransferase labeling in the presence of UDP-[3H]Gal. Top, autoradiographs of the gels stained with G250 seen in the bottom panels. Many O-GlcNAcylated low-molecular-weight proteins (<50 kDa) are observed in the Ribo preparation (arrowheads). No substrate (NS) and positive ovalbumin (Ovb) controls are included. Reactions containing all the components except galactosyltransferase (−GalT) are shown in the top right panel. (B) Ribosomal preparations from rat liver were obtained and labeled as in A. Partial pretreatment with commercial hexosaminidase (Ribo+Hex) decreases labeling by galactosyltransferase. (C) Fractions from polysome profiles (top panel) obtained from HepG2 cells growing under normal conditions were TCA precipitated, separated by electrophoresis, and subjected to immunoblot (IB) analysis with O-GlcNAc antibody (middle). A competition control obtained by pre-incubating the antibody with 1 M GlcNAc shows specificity of the labeling (bottom). Sedimentation was from left to right. Data shown are representative of at least three independent experiments.