In spite of various sources of NO, both NO donors and eNOS-generated NO blocked the 26S proteasome functionality and FTY720 shared the same pathway, indicating the essential role of NO in 26S proteasomes regulation which was truly suppressive. To the best of our knowledge, this is the first demonstration of NO-elicited effects on 26S proteasome functionality with a reporter cellular system as well as a reporter mouse model in vivo. Furthermore, this is also the first evidence for the connection of a metabolic/nutrient sensor between a vascular endothelial protective molecule and the quality control machinery for regulated protein turnover. Despite an intensive research effort, it remains uncertain how 26S proteasome functionality is regulated under physiological or pathological conditions. Given the well-established effects of NO on important cellular processes including proliferation and apoptosis, this radical gaseous molecule receives an increased appreciation for its potential role in 26S proteasome regulation. NO has been reported to suppress 26S proteasomes causing p53 accumulation/apoptosis in microphages or p21 accumulation in VSMC. Likewise, through suppression of the proteasomal degradation, NO Foretinib c-Met inhibitor maintains FLIP protein stability to prevent apoptosis in cultured human bronchial epithelial cells. Mechanism underlying the suppressive effect of NO on the 26S proteasome has not been completely elucidated but likely includes post-translational modification, e.g., S-nitrosylation of the 26S proteasomes in VSMC; transcription regulation, e.g., the decreased gene expression of PA28, a proteasome regulatory subunit, in vasculature; or involvement of other required mediators, e.g., 
a caspase 3, a GSK-3b for IRS-2 stability, or a Ser/Thr phosphatase. Since independent studies have shown a potential connection of OGT to gene transcriptional regulation and GSK, it would be interesting to explore whether OGT is involved in the suppressive effect of NO on 26S proteasomes as reported. However, the opposite results have been reported regarding the effect of NO on 26S proteasomes. It is yet unknown whether the discrepancy attributes to difference in cell types or the presence of extra reactive oxygen species, e.g., hydrogen peroxide, in some of the studies. What is clear is that a 26S proteasome reporter system has not been used in any studies on NO-mediated 26S proteasome functionality. Therefore, results demonstrated in the present study may help to clarify uncertainties or controversies concerning NO-exerted effects on 26S proteasome functionality in endothelial cells. Another novel aspect of this study was the demonstration of OGT and its connection to NO-mediated impacts on 26S proteasomes. The present study showed that NO functioned as a physiological suppressor of 26S proteasome functionality via an OGT-dependent mechanism involving O-GlcNAc modification, likely on proteasomal Rpt2 protein in vascular endothelial cells. OGlcNAcylation is the O-linked attachment of N-acetylglucosamine onto Ser/Thr residues of cytosolic and nuclear proteins, catalyzed by OGT. O-GlcNAcylation has been believed to be an important regulatory mechanism for signal transduction. Although mechanisms underlying OGT regulation are not well understood, OGT-mediated OGlcNAcylation has drawn increased attention. To date, more than 80 different proteins including transcription factors, kinases, phosphatases, cytoskeletal proteins, nuclear hormone receptors, nuclear pore proteins, signal transduction molecules, and actin regulatory proteins have been shown to undergo OGlcNAcylation. A proteomic study in fruit flies demonstrated that several proteins in the 26S proteasome can be extensively OGlcNAcylated. There is evidence that the 19S subunit can be subjected to O-GlcNAcylation with consequent 26S proteasome inhibition.