Figure 8.

OGA exhibits reduced catalytic activity when bound to FAS. U2OS cells stably overexpressing pcDNA3.1 (control) or pcDNA3.1 V5-FAS (test) were treated with vehicle (V) or H₂O₂ (2.5 mm, 2 h). An anti-V5 antibody was used to enrich V5-FAS from control and test NETN cell lysates (1.6 mg). n = 3. A and B, V5, OGA, and actin (loading/negative control) were detected by Western blotting. Western blots for each antibody were exposed for equal lengths of time. A, analysis of the inputs and unbound fractions. B, analysis of the bound fractions (31.25%). C, OGA activity was measured in the inputs and unbound fractions using 4MU-GlcNAc (1 mm). n = 3, two technical replicates per assay. Fluorescence values were converted to picomoles/min and then normalized to the pcDNA3.1 vehicle-treated sample. D, OGA activity was assessed in the input, bound (on-bead, 31.25%), and unbound fractions using 4MU-GlcNAc (1 mm). n = 3, two technical replicates per assay. Fluorescence values were converted to picomoles/min/μg using densitometric analysis of OGA in the input serial dilutions and the bound fractions, and then normalized to the pcDNA3.1 vehicle-treated sample. Only the data from FAS-overexpressing cells treated with H₂O₂ are shown, as OGA protein and activity was absent in the bound fraction of the other samples. Data are presented as the mean ± S.E. Significance was determined by RM-1ANOVA followed by Tukey's MCT, and differences were considered statistically significant at p ≤ 0.0001 (****). The migration of molecular mass (MW) markers is indicated.