Figure 6

OGT assay in biological samples. Assays were performed under varying OGT amount in different samples. The data were collected 20 min after starting. Pre-reaction of the OGT mediated transglycosylation was performed at room temperature for 30 min in 90 μL OGT reaction buffer consisting of UDP-GlcNAc (0.5 mM) and peptide (0.5 mM). Then, 10 μL reaction mix consisting of PEP (0.7 mM), PK (2 U), probe (2 μL), and the enzyme mix (2 μL) was added into the above solution. Fluorescence at 587 nm (E _x = 535 nm) was recorded in 15 min. (1) Purified C-654 (light gray); (2) total protein extracted from sonicated C-654 expressing BL21 solution (gray); (3) total protein extracted from mice brain tissue (heavy gray). The error bars indicate the range of standard deviation of the experimental data.