FIG. 5.

Proteasome inhibition and transcriptional regulation are integrated via O-GlcNAc (A) Degradation versus synthesis of GFP-degron in MCF10AT cells cotransfected with the indicated constructs. Increased O-GlcNAc levels were associated with increased GFP fluorescence. Degradation of the proteolytic GFP-degron plasmid was measured using a fluorimeter. (B) Degradation of Suc-LLVY-AMC chymotryptic proteolytic peptide substrate is impaired in the presence of increased O-GlcNAc levels. MCF10AT cells were transfected with the indicated constructs, and lysates were incubated with peptide. LLVY cleavage was measured using a fluorimeter. (C) Western blot of corepressors and coactivator proteins in MCF10AT cells retrovirally transfected with RNAi targeting OGT or NCOAT. (D) Densitometry was performed using ImageJ for the blots shown in panel C. (E) Western blot for ERα in MCF10AT cell lysates treated under the indicated conditions and with TSA to induce proteolytic clearance of ERα. (F to L) Results of real-time quantitative RT-PCR of nuclear hormone receptors ERα and PRA/B, coactivators SRC1 and SRC3/AIB1, and corepressor proteins NCoR and SMRT. Samples were normalized to GAPDH. For all experiments depicted, error bars represent standard errors of the means. CON, control.