Fig. 4.

O-GlcNAcylation of Crohn's-associated Nod2 variant regulates its half-life and NF-κB activity in cells. (A) Control cells do not express Myc-Nod2. 702 cells express a Nod2 variant associated with Crohn's disease. Cell lysates treated with and without Thiamet-G (1 µM, 4 h) were immunopurified with Myc antibody and blotted with anti-Myc or anti-O-GlcNAc antibodies. (B) Relative amount of CTD110.6 to Nod2 Myc was plotted from three independent experiments as the means + SD. (C) HEK293T-702 cells were incubated with 1 µM Thiamet-G or DMSO for 4 h, and before cycloheximide treatment (100 µg/mL) at the indicated time intervals. Nod2 was detected by anti-Myc western blotting. (D) Relative amount of Nod2 Myc to actin was plotted from three independent experiments as the means + SD. (E) Dual-luciferase assay performed on HEK293T transient-transfected with 0.1 ng Nod2 and 702 plasmids in the presence of 100 nM Thiamet-G or DMSO for 4 h. After 4 h cells were incubated with 20 µM MDP for 5 h, harvested, and tested for luciferase activity. *P < 0.05 was considered as significant.