Figure 1.

Oxidative stress increases OGT and OGA expression, OGA activity, and O-GlcNAc levels. U2OS cells were treated with vehicle (V) or H₂O₂ (2.5 mm, 1–3 h). n = 10, unless otherwise indicated. A, expression of OGT, OGA, and actin, as well as O-GlcNAc levels, was assessed in NETN lysates (5 μg) by Western blotting (WB). Protein load was assessed by total protein stain (colloidal Coomassie G-250) and by Western blotting (actin). Molecular mass (MW) markers are indicated. B, quantitation of actin normalized to total protein (G-250). C, quantitation of O-GlcNAc levels normalized to actin. D, quantitation of OGT expression normalized to actin. E, NETN lysates (5 μg) were assayed for OGT activity using [³H]UDP-GlcNAc (0.5 μCi) and CKII acceptor peptide (1 mm). n = 4, three technical replicates per assay. F, quantitation of OGA expression normalized to actin. G, NETN lysates (5 μg) were assayed for OGA activity using 4MU-GlcNAc (1 mm). n = 6, two technical replicates per assay. B–G, data are presented as the mean ± S.E. Significance was determined by RM-1ANOVA followed by Dunnett's MCT, and differences were considered statistically significant at p ≤ 0.05 (*), p ≤ 0.01 (**), and p ≤ 0.0001 (****).