Identification of Nitric Oxide as an Endogenous Inhibitor of 26S Proteasomes in Vascular Endothelial Cells
Figure 8
Upregulation of either O-GlcNAcylation or OGT suppresses 26S proteasome functionality.
The UbG76V-GFP-transfected HUVEC were treated with vehicle (medium) and glucosamine (5 mM) for indicated time up to 4h and followed by (A) quantifications of poly-Ub-GFP protein levels with an anti-GFP antibody and O-GlcNAc modification of Rpt2 with an anti-Rpt2 antibody (on WGA pull-down) in Western blot; (B) Western blotting of global O-GlcNAc modified proteins with an anti-O-GlcNAc antibody; (C) proteasomal chymotrypsin-like activity assay. (D) Overexpression of OGT not GFP increased both Rpt2 O-GlcNAcylation and the levels of reporter protein. (E) Chymotrypsin-like activity in GFP- and OGT-overexpressing cells. The blots shown were representative of 3 independent experiments with similar results. * represents p<0.05 vs the control (n = 3). An overlaid portion (less exposure) of each whole blot indicating Ub-GFP is presented. Ub-GFP, ubiquitin-green fluorescent protein; OGT, O-GlcNAc transferase; UDP-GlcNAc, Uridine diphosphate-GlcNAc; WGA, wheat germ agglutinin.