Journal: Journal of the American Chemical Society

Article Title: The Metabolic Chemical Reporter 6‑Azido-6-deoxy-glucose Further Reveals the Substrate Promiscuity of O ‑GlcNAc Transferase and Catalyzes the Discovery of Intracellular Protein Modification by O ‑Glucose

doi: 10.1021/jacs.7b13488

Figure Lengend Snippet: A significant fraction of 6AzGlc-dependent labeling is O-linked. (A) Known O-GlcNAcylated proteins are labeled by 6AzGlc. H1299 cells were treated with either Ac46AzGlc (200 μM) or DMSO for 16 h, followed by CuAAC with a cleavable alkyne-biotin tag. After enrichment on streptavidin beads, the labeled proteins were eluted and visualized by Western blotting. The nonglycosylated protein β-actin is a negative control. (B) A notable fraction of 6AzGlc-dependent signal is sensitive to β-elimination. NIH3T3 cells were treated with either Ac46AzGlc (200 μM) or DMSO vehicle for 16 h, followed by CuAAC with alkyne-biotin, separation by SDS-PAGE and transfer to a PVDF membrane. The indicated membranes were then treated for 24 h with either H2O or 55 mM NaOH before analysis by streptavidin or Western blotting. (C) 6AzGlc is not incorporated into N-linked glycans. NIH3T3 cells were treated with either Ac46AzGlc (200 μM) or DMSO vehicle for 16 h. The corresponding cell lysates were then incubated with either PNGase-F or H2O vehicle as indicated before CuAAC with alkyne TAMRA and analysis by in-gel fluorescence. A fraction of the treated lysate was separated before CuAAC and analyzed by Lectin blotting with Concanavalin A (ConA).

Article Snippet: The blots washed three times in TBST for 5 min and incubated with biotin-conjugated Concanavalin A (Vector Lab), diluted 1:1000 in TBST, for 1 h. The blot was then washed 3× with TBST for 10 min. Then the blot was then incubated with Strep-HRP at 1:1000 in blocking buffer for 1 h. After being washed 3× with TBST for 10 min, the blot was developed using ECL reagents.

Techniques: Labeling, Western Blot, Negative Control, SDS Page, Incubation, Fluorescence