Figure 3.

Cellular assays. (A) Western blot analysis of the inhibition of NleB1 glycosylation of TRADD by 100066N and 102644N in HEK293T cells. (B) MTT assays. Quantification of normalized MTT signal intensities as a function of 100066N and 102644N concentrations added to HEK293T cells for 24 h. (C) OGT assays. OGT was incubated with a recombinant OGT-peptide substrate in the presence of 100066N and 102644N and then subjected to immunoblotting using an anti-O-GlcNAc monoclonal antibody. (D) Bacterial growth assays. EHEC (left) and Salmonella (right) were cultured in the presence of the indicated compounds and growth was monitored as a function of time. (E) 100066N and 102644N reduce Salmonella replication in macrophages. Inhibitors (1 or 10 μM) were added to RAW 264.7 cells for 1 h prior to infection with 10⁶ CFUs of the indicated Salmonella strains. Colony counts were enumerated 24 h later. Asterisks indicate a significant difference (p < 0.05) from samples lacking inhibitors, as determined using one-way analysis of variance (ANOVA) with Dunnett's multiple comparisons tests.