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. 2014 Dec 8;290(4):2137–2149. doi: 10.1074/jbc.M114.598821

FIGURE 3.

FIGURE 3.

Stimulation of the hexosamine pathway increased O-GlcNAcylation of Notch1 EGF repeats. A, HEK293T cells were transfected to express N1EGF1–36-MycHis alone or together with EOGT. Transfected cells were incubated with 25 mm glucose (Glc), 10 mm glucosamine (GlcN), or 10 mm GlcNAc for 36 h. Myc-tagged N1EGF1–36-MycHis was immunoprecipitated (IP) from lysates using anti-Myc antibody and immunoblotted as indicated. The levels of O-GlcNAc modification on N1EGF1–36-MycHis quantitated by densitometric analyses of immunoblots are shown below. B, HEK293T cells transfected to express N1EGF1–36-MycHis and EOGT or untransfected cells were incubated with various concentrations of mannitol. After 36 h, cell lysates were subjected to immunoblot analysis as indicated. Quantification of O-GlcNAc modification on N1EGF1–36-MycHis is shown below. C, HEK293T cells expressing N1EGF1–36-MycHis alone or together with EOGT were incubated with 10 mm GlcN for 36 h. Immunostaining was performed using anti-O-GlcNAc (CTD110.6; red) antibody. GFP expression indicates transfected cells. Bar, 10 μm.