Figure 3.
hOGA Inhibitors GlcNAcstatins G and H Elevate O-GlcNAc Levels in HEK293 Cells
(A) HEK293 cells were incubated with GlcNAcstatins C, F, and G for 6 hr at a range of doses. Cellular O-GlcNAcylation levels were qualitatively analyzed using an O-GlcNAc antibody. Densitometric quantitation of single western blots (GlcNAcstatins C and F) and of three O-GlcNAc western blots (GlcNAcstatin G) reveals EC50 values of 20 nM for GlcNAcstatin C, 290 nM for GlcNAcstatin F, and 20 nM for GlcNAcstatin G.
(B) Immunostaining using an O-GlcNAc antibody shows an elevation of total O-GlcNAc modification (green) of HEK293 cells. Increased concentrations of GlcNAcstatins G and H induce hyper-O-GlcNAcylation. Dose-response curves of GlcNAcstatin treatments demonstrate the efficiency of the inhibition. Images were analyzed with the IN cell analyzer, and O-GlcNAc signal in each frame was normalized over the DAPI signal (blue). The scale bar represents 20 μm.
See also Figure S1.