Western blot analysis of O-GlcNAc was performed by loading 10 µg of HEK293T or C2C12 whole cell lysates onto a 4-20% Tris-HCl polyacrylamide gel. Proteins were transferred to a PVDF membrane and blocked with 5% BSA/TBST for at least 1 hour. Membranes were then probed with a mouse monoclonal antibody recognizing O-GlcNAc (Product # MA1-038) at a dilution of 1:1000 overnight at 4°C on a rocking platform. Membranes were then washed in TBS-0.1%Tween 20 and probed with a goat anti-mouse-HRP secondary antibody (Product # 31430) at a dilution of 1:15000 for at least one hour. Membranes were washed and chemiluminescent detection was performed using Super Signal West Dura (Product # 34075). The image is a Western blot with antibody Product # MA1-038 with untreated (lanes 1 and 3) versus treated (lanes 2 and 4) for O-GlcNAc enrichment.