Fig. 1.

pRB and p107 are O-GlcNAc modified in vitro. pRB, p107, and nucleoporin p62 (positive control for O-GlcNAc modification) were in vitro transcribed/translated (ITT) with radiolabeled methionine. a Following 1 h of ITT, lysates containing pRB, p107, or p62 were separated by WGA chromatography and O-GlcNAc modified proteins specifically eluted with free GlcNAc and counted by liquid scintillation. Inset is an autoradiogram of 5% of ITT reaction following SDS–PAGE (Cont. is no plasmid DNA added). pRb full-length product is marked with a gray arrow while p107 and p62 are marked in black. b Following 2.5 h of ITT in the presence or absence of the broad-based kinase inhibitor staurosporine (60nM final concentration), WGA chromatography and liquid scintillation counting were performed on p107 and p62. c Following 2.5 h of ITT, p107- or p62-containing lysates had a galactose added to terminal GlcNAc residues with β-1,4-galactosyltransferase. Extracts were then passed over the galactose binding lectin RCA and modified proteins eluted with free galactose and counted by liquid scintillation