Figure 5.

Defects in O-GlcNAc modification on SMAD4 T63 antagonize TGF-β signaling. (A) SBE reporter gene activity was downregulated in cells expressing SMAD4 T63V. MDA-MB-468 cells stably overexpressing SMAD4 WT or T63V were co-transfected with (CAGA)₁₂-Luc and CMV-Renilla and incubated with siOGT for 48 h. 5 ng/ml TGF-β was introduced for 24 h. Data are given as mean ± standard error (n = 3). The significance were annotated as *p < 0.05 and **p < 0.01. P values were calculated using Student’s t test. Relative reporter gene activities used for statistical analysis are presented in Supplementary Table S2. (B) Proposed model for the contribution of O-GlcNAc in the TGF-β signaling pathway by stabilizing SMAD4 proteins. O-GlcNAc modification on SMAD4 T63 inhibits SMAD4’s interaction with GSK-3β which promotes SMAD4 degradation by proteasomes. As a result, defects in SMAD4 T63 O-GlcNAc attenuated TGF-β-responsive SBE reporter gene activity.